CN101330935A - Isolation and cultivation of stem/progenitor cells from the amniotic membrane of umbilical cord and uses of cells differentiated therefrom - Google Patents

Isolation and cultivation of stem/progenitor cells from the amniotic membrane of umbilical cord and uses of cells differentiated therefrom Download PDF

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CN101330935A
CN101330935A CNA2006800475358A CN200680047535A CN101330935A CN 101330935 A CN101330935 A CN 101330935A CN A2006800475358 A CNA2006800475358 A CN A2006800475358A CN 200680047535 A CN200680047535 A CN 200680047535A CN 101330935 A CN101330935 A CN 101330935A
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T-T·潘
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CELLRES CORP Pte Ltd
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Abstract

The present invention relates to a skin equivalent and a method for producing the same, wherein the skin equivalent comprises a scaffold and stem/progenitor cells isolated from the amniotic membrane of umbilical cord. These stem/progenitor cells may be mesenchymal (UCMC) and/or epithelial (UCEC) stem cells, which may then be further differentiated to fibroblast and keratinocytes. Further described is a method for isolating stem/progenitor cells from the amniotic membrane of umbilical cord, wherein the method comprises separating the amniotic membrane from the other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions allowing cell proliferation, and isolating the stem/progenitor cells from the tissue cultures. The invention also refers to therapeutic uses of these skin equivalents. Another aspect of the invention relates to the generation of a mucin-producing cell using stem/progenitor cells obtained from the amniotic membrane of umbilical cord and therapeutic uses of such mucin-producing cells. In yet another aspect, the invention relates to a method for generating an insulin-producing cell using stem/progenitor cells isolated from the amniotic membrane of umbilical cord and therapeutic uses thereof. The invention further refers to a method of treating a bone or cartilage disorder using UCMC. Furthermore, the invention refers to a method of generating a dopamin and tyrosin hydroxylase as well as a HLA-G and hepatocytes using UCMC and/or UCEC. The present invention also refers to a method of inducing proliferation of aged keratinocytes using UCMC.

Description

The application that separates and cultivate the cell of ancestral cells and differentiation thereof from the umbilical cord amniotic membrane
The intersection document of related application
The present invention requires to enjoy in the priority of the U.S. Provisional Application submitted on October 21st, 2005 number 60/729,172, and its content quotes in full herein as a reference with it for all purposes.
FIELD OF THE INVENTION
The method that the present invention relates to the skin equivalent and produce this skin equivalent, wherein the skin equivalent comprises support and from the isolating ancestral cells of umbilical cord amniotic membrane.These ancestral cells can be mesenchyme (UCMC) and/or epithelium (UCEC) stem cell, and it can also further be divided into fibroblast and keratinocyte.What further describe is the method for isolation of stem/progenitor cells from amniotic membrane of umbilical cord, and wherein method is included in and external amniotic membrane other composition with umbilical cord is separated, and cultivates amnion tissue under the condition that allows cell proliferation, and isolation of stem/progenitor cells in the self-organizing culture.The invention still further relates to the therapeutic use of these skin equivalents.Another aspect of the present invention relates to use and produces mucoprotein-production cell and the therapeutic use of this mucoprotein-production cell from the ancestral cells of umbilical cord amniotic membrane acquisition.In yet another aspect, the present invention relates to use from the isolating ancestral cells generation of umbilical cord amniotic membrane insulin-production cell and therapeutic use thereof.The invention still further relates to the method for using UCMC treatment bone or cartilage disease.In addition, the present invention relates to use UCMC and/or UCEC to produce dopamine and tyrosine hydroxylase and HLA-G and hepatocellular method.The invention still further relates to and use UCMC to induce the method for old and feeble keratinocyte propagation.
Background of invention
Stem cell is the cell colony that has unlimited self renewal and be divided into the ability of various kinds of cell type or types of organization.Embryonic stem cell (after fertilization was from about 3 to 5 days) infinite multiplication and can be divided into whole types of organizations natively: therefore claim that they are that myeloid-lymphoid stem cell (pluripotent stem cell) (is seen and summarized for example Smith, A.G. (2001) Annu.Rev.Cell.Dev.Biol.17,435-462).Yet, adult stem cell has more tissue specificities and may have relatively poor fertility: therefore claim that they are that pluripotent stem cell (multipotent stem cell) (is seen and summarized for example Paul, (2002) DrugDiscov.Today 7 such as G., 295-302)." plasticity " of embryonic stem cell and adult stem cell depends on them changes the ability different with its origin and that may cross over the tissue of embryonic germ layer that is divided into.
The ability of stem cell self renewal is critical to them as the function of original undifferentiated cell depots.On the contrary because telomere shortens, most of somatic cell have limited self renewal ability (see and summarize for example Dice, J.F. (1993) Physiol.Rev.73,149-159).Therefore, the therapy based on stem cell has the potentiality that are used for the treatment of the multiple disease of humans and animals.
Stem cell and ancestral cells can be derived from separate sources." multispectral system " potential of embryonic stem cell and adult stem cell is extensively characterized.Although the potential of embryonic stem cell is huge, their purposes causes many ethical issuess.Therefore, suggestion will be originated as an alternative derived from the non-embryonic stem cells of bone marrow matrix, fatty tissue, corium and Cord blood.These cells especially can vitro differentiation be chondrocyte, adipose cell, osteoblast, sarcoplast, myocardial cell, astrocyte and Tenocyte cell and in vivo (in vivo) break up, make the stem cell of these so-called mescenchymal stem cells become the promising candidate target that is used for mesoderm defect repair and disease treatment.
Yet in clinical use, gather in the crops this type of mescenchymal stem cell and cause some problems.Because need surgical method, so the collection of cell causes mind ﹠ body burden (for example, the collection of bone marrow is implemented with biopsy needle, needs the invasive technique of part even general anesthesia) to patient so that obtain cell.In addition, in many cases, the stem cell number of extraction is quite few.The more important thing is that these cells are not derived or are divided into epithelial cell.This has promoted to search the stem cell that other may be originated.
Differentiated that Cord blood can be used as the abundant source of hematopoietic stem.Yet, whether the existence of mesenchyme ancestral cells is still had arguement.In one aspect, this type of cell can not in mature Cord blood, separate or successfully cultivate (Mareschi, K. etc. (2001) Haematologica 86,1099-1100).Meanwhile, by Campagnoli, C. etc. (Blood (2001) 98,2396-2402) and Erices, A. etc. (Br.J.Haematol. (2000) 109, the results suggest that 235-242) obtains, mescenchymal stem cell are present in some fetus organs, and circulate simultaneously with the hemopoietic precursor in the blood of prematurity of fetus.Therefore, International Patent Application WO 03/070922 discloses from the Cord blood separation and has cultivated the method for amplification mesenchyme ancestral cells and make this type of cell differentiation become the method for multiple mescenchymal tissue.It is reported, separation efficiency be about 60% (Bieback, K. etc. (2004) Stem Cells 22,625-634).In same research, determine, be the important parameter of realizing this productive rate from collecting Cord blood to the time period of isolated cell and the volume of used blood sample.Yet whether these ancestral cells are derived from umbilical cord tissue really still exists arguement.
Recently, from umbilical cord tissue, i.e. successful separating mesenchymal ancestral cells (Mitchell, K.E. etc. (2003) Stem cells 21,50-60 from the umbilical cord matrix jelly of Wharton; United States Patent (USP) 5,919,702; U.S. Patent application 2004/0136967).Having shown that these cells have is divided into for example ability of neuron phenotype and cartilaginous tissue respectively.In addition, the umbilical vein endodermis of one of three blood vessels that also existed in umbilical cord (two radicular arterieses, a radicular vein) has separated mesenchyme ancestral cells (Romanov, Y.A. etc. (2003) Stem cells 21,105-110 with subendothelial layer; Covas, D.T. etc. (2003) Braz.J.Med.Biol.Res.36,1179-1183).
Yet these used so far methods all do not obtain the separation or the cultivation of epithelium ancestral cells, and described epithelium ancestral cells is the source as the epithelial cell treatment, the surface texture of for example skin shaping, liver reparation, bladder body engineering and other through engineering approaches.The skin shaping is a therapeutic treatment crucial especially and that press for, and for example from the obtainable number of the U.S., it still needs a large amount of exploitations.Only, the burn and 600, the 000 example operation skin excisions of 100,000 hospitalizes just there is every year in the U.S..The problem of old and feeble disunion corium wound of being correlated with is more, in the U.S. 1,100 ten thousand to 1,200 ten thousand subject patients is arranged.For these diseases, Europe shows patient's number about equally.
Skin has three layers, epidermis, corium and fat deposit, and it all carries out specific task.Epidermis is mainly produced by the keratinocyte of epithelial origin, and corium is to be assembled by the fibroblast that mesenchyme is originated to form.Epidermis is thin, the tough and tensile top layer of skin.The outer part of epidermis, horny layer is a waterproof, and when not damaging, can prevent that most of antibacterial, virus and other foreign substance from entering body.Epidermis also protects internal, muscle, nerve and blood vessel to avoid wound.Epidermis also contains islet cells, and it is the part of skin immune system.Following one deck---the corium of skin is the thick-layer (major part is made of polymer collagen and FBN1) of fiber and Elastic tissue, produces the elasticity and the intensity of skin.Corium contains teleneuron, body of gland, hair follicle and blood vessel.
Because mechanical force requires the skin replacement usually as scratch, the operation wound relevant with skin carcinoma excision or owing to burn or the damage of epidermis that skin loss that other wounds such as chronic venous ulcer cause etc. causes, corium or two-layer (holostrome wound).According to the degree of damage,, be not enough sometimes because the extensive disappearance of skin is replaced with patient self skin (autograft) of taking from the unaffected part of body.Therefore, there is help, the needs of the substitute of research and development damaged skin by autogenous cell or homogeneous variant cell.
For example, U.S. Patent number 6,479,875 have described Graftskin, and it is made up of support, and described support has been integrated the corium be made up of mescenchymal stem cell-form cell.Yet these are rare from the isolating mescenchymal stem cell of bone marrow, in order to obtain enough mescenchymal stem cells, must obtain the 10-20cc aspirate from the patient usually.
Therefore, still need to be used to separate and cultivate the method and the reliable sources of epithelium ancestral cells, it can be used for the further suitable Graftskin of research and development.In addition, still need can accept ethically, and the patient do not caused biomedical burden, be used to separate epithelium and mesenchyme ancestral cells fast and effective method so that provide capacity this type of cell for the multiple application of regenerative medicine and organizational project.
Summary of the invention
The invention provides the skin equivalent that comprises support, described support comprises from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane.
In one embodiment of the invention, be mescenchymal stem cell (UCMC: in other words mean umbilical cord liner (amniotic membrane) mescenchymal stem cell from the deutero-cell of ancestral cells; Be also referred to as CLMC) or epithelial stem cell (UCEC: in other words mean umbilical cord liner (amniotic membrane) epithelial stem cell; Be also referred to as CLEC).
The cell that is used for skin equivalent of the present invention can be from body, xenogenesis or allochthonous cell.
In addition, the cell that is used for skin equivalent of the present invention can be the mammal source.In one embodiment of the invention, cell is a human origin.
In another embodiment, the skeleton of skin equivalent can also comprise other cell line, such as but not limited to, vascular endothelial cell or microvascular dermal endothelial cell.In one embodiment, these vascular endothelial cells are derived from umbilical cord.According to donor, endotheliocyte can be mammal or human origin.
In one embodiment of the invention, the skin equivalent comprises support, and it comprises biological degradable material.In another embodiment, this support comprises or is made up of following material, but be not limited to these materials, for example agarose, polycaprolactone, niobium bag are by carbon, chitosan, collagen, hyaluronic acid, calcium phosphate, starch, hydroxyapatite, fibrin, alginate, polyglycolic acid, carbon nano-fiber (carbon nano fibres), porous Merlon, politef, polylactide (polylactide) and composition thereof.In an example of the present invention, timbering material is a Merlon.
The present invention also provides support, and this support can comprise that at least a extracellular matrix is as the holder from the deutero-cell of the isolating ancestral cells of the amniotic membrane of umbilical cord.Extracellular matrix component can include but not limited to one or more materials, for example collagen, elastin laminin, intercellular adhesion molecule, laminin, heparin, fibronectin, Dan Baijutang, tenascin, FBN1 and composition thereof.In one embodiment, extracellular matrix component is a collagen.In another embodiment, extracellular matrix be by ancestral cells self by the secretion separately extracellular matrix component for example collagen provide.
In another embodiment of the invention, UCMC that comprises in skin equivalent of the present invention and/or UCEC can breed and further be divided into fibroblast and keratinocyte respectively.
The present invention also provides the method for producing the skin equivalent, and it comprises:
● support is provided,
● will from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane be placed in the described support or on the support and
● hatch described support in first culture medium, it allows described cell proliferation and further differentiation.
In one embodiment, be mescenchymal stem cell (UCMC) or epithelial stem cell (UCEC) from the deutero-described cell of ancestral cells.
In another embodiment of the invention, when described support comprised UCMC and UCEC, described first culture medium comprised the culture medium that is suitable for fibroblast or keratinocyte cultivation respectively.
The present invention also provides the method for treatment dermatosis, comprises with skin equivalent of the present invention contacting described dermatosis.The present invention also provides skin equivalent of the present invention or is used for the application of the production of pharmaceutical composition by the skin equivalent that method of the present invention obtains, and pharmaceutical composition is used for the treatment of the skin of burn and the application of ulcer, only enumerates the example of some exemplary dermatosis.
The present invention also provides the cell bank of the skin equivalent that comprises skin equivalent of the present invention or obtain by method of the present invention.
The present invention also is provided for producing the method for mucoprotein-production cell, comprising:
● umbilical cord amniotic membrane inner lining film epithelium or mescenchymal stem cell (being respectively UCEC and UCMC) be placed in the container and
● in the culture medium that is fit to the cultivation secretory cell, hatch described UCEC or UCMC.This mucoid-production cell for example can be used for treating the ocular surface that influenced by cigarette or the cell of respiratory tract.
In another embodiment, the present invention also provides the method for generation insulin-production cell, comprises
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane and
● in proper culture medium propagation with break up described cell and become β-islet cells.
In another embodiment, the invention provides insulin-production cell by the method acquisition of the invention described above, and treatment and the unbalance relevant disease of insulin level, comprise the insulin production cell that obtains by above-mentioned method of the present invention to administration.
In another embodiment of the invention, the method for treatment osteopathia is provided, comprise to the patient and use the osteoblast that produces by mescenchymal stem cell that described mescenchymal stem cell separates from umbilical cord amniotic membrane (UCMC).The method of treatment cartilage disease also is provided, comprises to the patient and use chondrocyte that it is by producing from the isolating mescenchymal stem cell of umbilical cord amniotic membrane (UCMC).
Another embodiment of the invention provides the method that produces dopamine and tyrosine hydroxylase production cell, comprises
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane, preferred UCMC and
● in proper culture medium propagation with break up described cell and become dopamine and tyrosine hydroxylase to produce cell.
In addition, the present invention relates to use the method for UCMC and UCEC production Leucocyte Antigen G respectively (HLA-G) or liver like cell.
In another embodiment of the invention, relate to the method for inducing old and feeble keratinocyte propagation, comprise
● in suitable growth medium, cultivate old and feeble keratinocyte, and in the keratinocyte of aging, add the mescenchymal stem cell (UCMC) of umbilical cord amniotic membrane, thereby induce the propagation of old and feeble keratinocyte.
Brief description
When considering limiting examples and accompanying drawing simultaneously, will understand the present invention better with reference to detailed description, in the accompanying drawings:
Fig. 1 has described by directly organizing the explant method, the epithelial cell growth from the umbilical cord amniotic membrane dizzy (40 * amplification) in tissue culture the 2nd day (Figure 1A) and the 5th day (Figure 1B, C).Before amniotic membrane is positioned over the surface, with or need not 1 Collagen Type VI/4 Collagen Type VI mixture (1: 2; Becton Dickinson and Co. (Becton Dickinson), New Jersey, USA) coated cell is cultivated frosting.The amniotic membrane specimen be immersed in 5ml EpiLife culture medium or culture medium 171 (both are all from Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA) in.Changed culture medium in per 2 or 3 days, the cell outgrowth of monitoring explant under optical microscope.Take microphotograph at different intervals as mentioned above.Observed polyhedral cell form is typical epithelial cell.
Fig. 2 describes the segmental enzymatic digestion of umbilical cord, produces similar epithelial cell (40 * amplification) at the 2nd day (figure A, C) and the 5th day (figure B, D).The umbilical cord amniotic membrane is divided into the small pieces of 0.5cm * 0.5cm, 0.1% (w/v) 1 Collagen Type VI enzymatic solution (F. Huffman-La Luoqie company (F.Hoffmann-Laroche Ltd.) Basel, Switzerland) in 37 ℃ of digestion 8 hours.Per 30 minutes vortex mixed of sample 3 minutes.At centrifugal 30 minutes collecting cells of 4000rpm.Cell precipitation is resuspended in adds 50 μ g/ml insulin-like growth factor-is (IGF-1), 50 μ g/ml platelet derived growth factor-BB (PDGF-BB), 5 μ g/ml transforming growth factor-beta (TGF-β 1) and 5 μ g/ml insulins (all from R﹠amp; (the R﹠amp of d system company; DSystems), Minneapolis, USA obtains) the EpiLife culture medium or culture medium 171 (both are all from Cascade Biologies) in, counting is also by 1 * 10 6The density of individual cell/ware is inoculated into 1 Collagen Type VI/4 Collagen Type VI mixture (1: 2; Becton Dickinson and Co. (Becton Dickinson), New Jersey USA) wraps 10 cm tissue culture wares of quilt in advance.After 24 hours, the cell that adheres to the washing of the saline solution (PBS) of warm phosphoric acid buffer, and with EpiLife culture medium or culture medium 171 replacement culture medium (both are all from Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA).Change a subculture in per 2 or 3 days, and under optical microscope, monitor the cell outgrowth.Take microphotograph at different intervals as mentioned above.Cell demonstrates the polygonal form of typical epithelial cell once more.
Fig. 3 describes the mesenchymal cell that grows out from the outer planting of umbilical cord amniotic membrane.Use was added the DMEM of 10% tire calf serum (FCS) as culture medium, early observed cell outgrowth (40 * amplification) (Fig. 3 A, C) after placing tissue culture's ware to 48 hours.With explant be immersed in 5ml add 10% hyclone (U.S. Hyclone company (Hyclone), Utah, DMEM USA) (hero Life Technologies, Inc. (Invitrogen Corporation), California, USA) in (DMEM/10%FBS).Culture medium was changed once in per 2 days or 3 days.Monitoring cell outgrowth under optical microscope.Take microphotograph at different intervals.Cell have the spindle form and external easily and the feature of moving fast and stretching, very similar fibroblast (Fig. 3 B, D).
Fig. 4 (40 * amplify) describe by the collagenase enzymatic digestion, from the isolating mesenchymal cell of umbilical cord amniotic membrane.Fig. 4 A is presented at the 2nd day from the isolating mesenchymal cell of umbilical cord amniotic membrane.Observed cell proliferation (Fig. 4 B) at the 5th day.The umbilical cord amniotic membrane is divided into the small pieces of 0.5cm * 0.5cm, and 0.1% (w/v) 1 Collagen Type VI enzymatic solution (F. Huffman-La Luoqie company (F.Hoffmann-Laroche Ltd.) Basel, Switzerland) in 37 ℃ of digestion 6 hours.With per 15 minutes vortex mixed of sample 2 minutes.By centrifugal 30 minutes collecting cells of 4000rpm.Cell precipitation is resuspended among the DMEM/10%FBS, and counting also presses 1 * 10 6The density of individual cell/ware is seeded in the 10cm tissue culture ware.Culture medium was changed once in per 2 days or 3 days.Monitoring cell outgrowth under optical microscope.Take microphotograph at different intervals.Cell demonstrates once more as the typical spindle form of fibroblastic mesenchymal cell.
Fig. 5 (40 * amplify) be described under serum-free culture condition (DMEM) and the serum condition of culture (DMEM/10%FCS) according to the isolating umbilical cord amniotic mesenchymal cell of the inventive method (UCMC, Fig. 5 E, F, G, H), normal dermal fibroblast (NF109 cell, Fig. 5 A, B) and the form of adipose-derived mesenchymal cell (ADMC, Fig. 5 C, D).Serum rich conditions (DMEM/10%FCS) is compared in Fig. 5 demonstration, NF that cultivates under serum starvation condition (DMEM is only arranged) and the cellular morphology of ADMC change, it shows as more flat cell and more unsound Cytoplasm, and wherein cell is round and have a fine and close Cytoplasm (Fig. 5 A, B, C, D).In two groups of UCMC groups of under the serum-free of the same terms and serum rich medium, cultivating, do not observe morphologic change (Fig. 5 E, F, G, H), show that these mesenchymal cells of back are in behavior and physiological difference.
Fig. 6 (40 * amplify) has described the isolating UCMC according to the present invention, and it is when no 3T3 feeder layer, the 3rd day and the 7th day UCMC cultivating in DMEM/10%FCS.The finding cell is the cell of growing, and is forming colony (vertical-growth) and do not show radial extension.This shows that again these mesenchymal cells are compared the difference in behavior with the corresponding cell that it more breaks up.
The colony that Fig. 7 (40 * amplification) has described the umbilical cord epithelial cell (UCEC) of cultivating the 3rd and the 7th day on the 3T3 feeder layer forms.This outward appearance is similar to the outward appearance of the normal deutero-epithelium keratinocyte of skin stem cell.In the latter, the 3T3 feeder layer is kept the stem cell property (stemness) of cell.
Fig. 8 (40 * amplify) the significantly colony formation (Fig. 8-1) of isolating umbilical cord mesenchyma cell (UCMC) according to the present invention was described the 3rd and the 7th day that cultivates on the 3T3 feeder layer.The 3T3 feeder layer suppresses the growth of the mesenchymal cell that breaks up usually, as the human dermis fibroblast.This corresponding cell that shows that again these mesenchymal cells and its more break up is compared the difference in behavior.Fig. 8-2 has shown that the colony of umbilical cord amnion cell forms efficiency test.
Fig. 9-1 shows the Western engram analysis to Fig. 9-28, compared expression and the expression in human dermis fibroblast (NF), medulla mesenchyma cell (BMSC) and adipose-derived mesenchymal cell (ADMC) of several embryonic stem cell marks in isolating UCEC and UCMC according to the present invention by this analysis.Fig. 9-29 shown with stem cell, human dermis fibroblast and the epithelium keratinocyte of bone marrow, adipose-derived with 9-30 and compared, and the secretion (Fig. 9-29) of the leukaemia inhibitory factor that the Western engram analysis detects and ELISA measure the high excretory activator protein A of detection and Follistatin (Fig. 9-30) respectively in the supernatant of umbilical cord amniotic membrane and epithelial stem cell culture.The fixing colony in the culture dish, and, confirm the expression (Fig. 9 .1.b) of transcription factor with anti-Oct-4 antibody (Fig. 9 .1b) dyeing.
Figure 10 is presented at the indirect immunofluorescence analysis of the epithelial cell mark of expressing in the umbilical cord epithelial stem cell, for example total cytokeratin (CK), CK17, CK6, CK10, CK19, CK18, CK16, CK15 (Figure 10-1); Hemi desmosome composition-integrin alpha 6, the plain β 4 of integration; Desmosome composition (Figure 10-2); Basement membrane components-laminin 1, laminin 5, IV Collagen Type VI, VII Collagen Type VI (Figure 10-3) and other important extracellular matrix composition are as integrating plain β 1 and fibronectin (Figure 10-4).
The cytokine array analysis by excretory cytokine of umbilical cord mesenchymal stem cells (UCMC) and somatomedin is compared in Figure 11 demonstration with human marrow mesenchymal stem cell.
The cytokine array analysis by excretory cytokine of umbilical cord epithelial stem cell (UCEC) and somatomedin is compared in Figure 12 demonstration with the human epidermic keratinocyte.
Figure 13 is presented at the DMEM (Figure 13-1) that adds 10% tire calf serum (FCS), at serum-free medium PTT-1 (Figure 13-2), at serum-free medium PTT-2 (Figure 13-3, Figure 13-4) and the UCMC cell in serum-free medium PTT-3 (Figure 13-5), cultivated.Figure 13 also is presented at the growth of adipose-derived stromal cell among the serum-free medium PTT-3 (Figure 13-6) and derived from bone marrow stromal cell (Figure 13-7).
Figure 14 has shown by the umbilical cord epithelial stem cell of dna microarray analysis and comprehensive gene expression of mescenchymal stem cell.UCEC expresses altogether 28055 genes and UCMC expresses 34407 genes altogether.In two kinds of cell types, express 27308 eclipsed genes.747 gene pairs UCEC that express are unique, and 7099 gene pairs UCMC that express are unique.In this figure, list selected genes of interest.Two types stem cell is all expressed 140 genes relevant with embryonic stem cell and fetal development.
Figure 15 shows that the repetition explant that uses umbilical cord inner lining film tissue expands the sketch map of umbilical cord epithelial stem cell and mescenchymal stem cell.
Figure 16 describes the cross section of umbilical cord, shows two umbilical arterys (UA) and the umbilical vein (UV) that support in umbilical cord amniotic membrane inner lining film (LM), the jelly of Wharton (WJ) that comprises and the jelly of Wharton.
Figure 17 describes and directly to be divided into skin epithelium keratinocyte (Figure 17 A) from the isolating epithelial cell of umbilical cord amniotic membrane (UCEC), and becomes osteoblast (Figure 17 B) and adipose cell (Figure 17 C) from the isolating mesenchymal cell of umbilical cord amniotic membrane (UCMC) in vitro differentiation.
It is skin epithelium keratinocyte (Figure 18 a that Figure 18 has described from the isolating epithelial cell of umbilical cord amniotic membrane inner lining film (UCEC) vitro differentiation; The photo of taking after 7 days at cell culture), with become fibroblast (Figure 18 b from the isolating mesenchymal cell of umbilical cord amniotic membrane inner lining film (UCMC) in vitro differentiation; The photo of taking after 7 days at cell culture).
Figure 19 (200 * amplify) has described the skin of the comprehensive growth that obtains by method of the present invention etc. and has hindered thing.Epithelial layer is made of keratinocyte, and described keratinocyte is to hatch in the concrete culture medium that limits of the inventive method and break up generation by UCEC.Skin corium is to hatch in the concrete culture medium that limits of the inventive method and the keratinocyte that breaks up generation constitutes by UCMC, also is grown in the outer collagen stroma of born of the same parents, and is included within the skin equivalent of the present invention.
Figure 20 a (1200 * amplification) has described the keratinocyte appearance of the skin equivalent (CSE-1) of the method generation of describing according to embodiment 12.Figure 20 b (2000 * amplify) outward appearance of the deutero-fibroblast of UCMC in collagen scaffold (grid) described, described cell is to obtain according to the method that embodiment 12 describes.
Figure 21 a (2000 * amplification) has described the keratinocyte appearance of the skin equivalent (CSE-2) of the method generation of describing according to embodiment 13.Figure 21 b (3000 * amplify) outward appearance of the deutero-fibroblast of UCMC in collagen lattice described, described cell is to obtain according to the method that embodiment 13 describes.
Figure 22 described of the present invention mucoprotein-produce cell.Figure 22 a has shown cultivated mucoprotein after 3,7 and 10 days-production cells whose development in PTT-6.Figure 22 b has shown that the UCEC that cultivates ( precipitation 1,2,3,6 (P1, P2 etc.) that refers to UCEC-17) that detects by its molecular weight in SDS-PAGE produces in PTT-6 mucoprotein.Other details is referring to embodiment 16.
Figure 23 has described the UCEC that is incubated in PTT-10 and the nicotiamide.As from photo as seen, the UCEC of hatching with nicotiamide is divided into β-islet cells (Figure 23 b), the UCEC that grows in having only PTT-10 does not have then that (Figure 23 a).
Figure 24 has described for cartilage development, and the UCMC cartilage forms and is divided into chondrocyte.Based on inducing, used alcian blue (Alcian Blue) dyeing from the chondrocyte that UCMC grows with the PTT-5 that modifies.In Figure 24 B, observe the positive staining of chondrocyte.Undifferentiated UCMC cell observation is to negative staining (Figure 24 A).
Figure 25 has shown the insulin expression in a plurality of UCEC samples, described UCEC is in ES Cult culture medium (Stemcell Technologies Inc. (CA) (Stem Cell TechnologiesInc.) of describing among the embodiment 15, Vancouver is Canada) or under the inducing of BBRC06 culture medium.This tests demonstration, and UCEC has the potential that is divided into insulin-production cell, and it can be used for treating diabetes.
Figure 26 A and Figure 26 B have showed that tyrosine hydroxylase (TH) and dopamine pass through the secretion and the expression of the UCMC cell of differentiation, and more detailed description is in embodiment 18.Dopamine is used for the treatment of the parkinson's syndrome patient.Figure 26 C shows negative control.
Figure 27 A and Figure 27 B have showed that the HLA-G that describes among the embodiment 19 is by the UCMC of differentiation and the secretion and the expression of UCEC cell.
Figure 28 A, 28B and 28C show that UCMC induces old and feeble skin keratin to form the result of experiment of cell (asK) and human dermis's fibroblasts proliferation.This experiment is used from 50 or the Skin Cell that obtains of 60 years old patient.This experiment shows the cultivation effect of UCMC, therefore it also can be used for wound healing, tissue repair, regeneration, rejuvenation (rejenuvation), cosmetics and skin care applications.
Figure 29 A has shown that UCMC and the UCEC organotypic in collagen lattice cultivates altogether.In these mescenchymal tissue equivalent (MTE) constructs, observed epithelium.Figure 29 B shows the skin similar structures of cultured skin equivalent.The differentiation fully of the collagen lattice backer keratinocyte stem cell of UCMC cell proliferation.These accompanying drawings show that UCMC and UCEC can be used for the outer organ sample tissue of construct, are used for tissue repair and regeneration and drug discovery.
Figure 30 A and 30B have showed that the UCMC cell can be grown in TissueFleece
Figure A20068004753500231
E (Austria hundred special companies (Baxter AG), Austria) and BoneSave
Figure A20068004753500232
(U.S. Stryker Corp. (Stryker Inc.), MI, within collagen USA) and the bone support and on.Accompanying drawing shows the UCMC alive on the support that is colored, be grown in embodiment 2 descriptions.
Figure 31 refers to the experiment that embodiment 22 describes, and has showed the angiogenic character of the collagen scaffold of the UCMC gathering (populated) of implanting mice.Figure 31 B and C show the vasculation of the both macro and micro of observing after 21 days.Figure 31 A shows the preceding accumulative support of UCMC in growth medium of implantation.
Figure 32 A to C shows that UCMC is used for the treatment of the clinical practice of FTB (3 degree).The wound bed that Figure 32 A is presented on 53 years old old women patient's the FTB is prepared.With the UCMC cell inoculation to the Biobrane wound dressing (but her pharmaceutical companies (Dow Hickam Pharmaceuticals) of Tao Shi, Texas, USA) on.By the description among the embodiment 23 the UCMC-Biobrane construct is transferred to (Figure 32 B) on the wound.Do not having dermatoplastic the 7th day to observe healing fully, and following up a case by regular visits to stable (Figure 32 C) in 3 months.
Figure 33 has showed that UCMC is used for the treatment of the clinical practice of the 2 years old male patient's who describes among the embodiment 24 partial-thickness wound (2 degree).Observed the healing fully of wound at the 3rd day.
Figure 34 has showed that UCMC is used for the treatment of the clinical practice of 2 years old male patient's FTB (3 degree).With UCMC and SoloSite
Figure A20068004753500233
The glue ((Smith﹠amp of brightness company of Xerox; Nephew), Hull UK) mixes, and the paste of pressing description among the embodiment 23 is on wound.Observed the healing fully of wound at the 5th day with this method.
Figure 35 has showed that UCMC is used for the treatment of the clinical practice of the non-healing radiation wound of suffering from angiomatous 1 years old child.With the trauma care of routine, primary trauma was not healed 90 days period.UCMC is at Tegaderm
Figure A20068004753500234
Cultivate in the wound dressing and transferring on the wound as description among the embodiment 25.Through 20 days by a definite date UCMC cell therapy, the radiation wound healed fully.
Figure 36 and Figure 37 A and B have showed that UCMC is used for the treatment of the clinical practice of the flap of non-healing diabetic wound (Figure 36), non-healing dieletic foodstuff wound (Figure 37 B) and failure for district's wound (Figure 37 A).Both can not heal the back through the conventional therapy above 6 years.UCMC and and SoloSite are cultivated in the description of pressing among the embodiment 26
Figure A20068004753500241
The glue ((Smith﹠amp of brightness company of Xerox; Nephew), Hull UK) mixes.
Figure 38 is presented at the BBRC06H culture medium, and (the BBRC06H culture medium is the modification version of BBRC06, as describing among the embodiment 15, does not add nicotiamide and adds the inducing albumin expression of UCEC down of the Oncostatin. of 50 μ g/ml-M).This experiment shows that UCEC has and is divided into hepatocellular potentiality that it can be used for treating hepatic disease or is used to test the Cytotoxic external model of new drug.
In detail explanation
The present invention is based on astonishing discovery, namely can use the umbilical cord amnion to form the skin equivalent as the source, can be under vitro conditions from described source successful separation and the ancestral cells that increases, for example between mesenchymal ancestral cells and epithelium ancestral cells. Use these cells, invention provides the skin equivalent, and it comprises or substantially is comprised of support, and described support comprises the cell from the ancestral cells of deriving from the umbilical cord amnion. These ancestral cells performance embryonic stem cell sample features of more surprised discovery. Recently, amnion (being also referred to as the amnion inner lining film), namely wrap up placenta and grow the inner layer film capsule of mammal embryo, use (reference example such as Anderson as the natural substrate of eye surface reconstruction and the biological substrate of amplification corneal limbal epithelial stem cell, (2001) the Br. J.Ophthalmol.85 such as D.F., 567-575 Gr ü terich, M. etc. (2003) Surv.Ophthalmol.48,631-646). Yet, also do not have so far to describe the method that is used for from the amnion isolation of stem/progenitor cells, at least for the mankind, do not report the amnion that will cover umbilical cord as source of human stem cell yet, described stem cell is for the production of skin equivalent of the present invention.
Support is used as the substrate of skin equivalent of the present invention. Field of tissue engineering technology has been used widely support to make up and can have been transplanted the new organization of repairing the defective position in the body, or in the bio-artificial device as cell container. Support forms three dimensional matrix, as cell propagation and the final template that forms of organizing. In support, cultivate cell and be usually directed at whole support repopulating cell, and allow cell in support, to breed the time of scheduled volume.
Therefore, the invention provides skin equivalent and its method of acquisition, wherein in one embodiment, support comprises or is made by biodegradable material. It is favourable that support uses biodegradable material, and for example, for organizational project, the support that wherein contains cell is used to repair the defective position in living tissue such as the skin. An effective aspect of the support that uses among the present invention be they for the penetrating property of cell culture medium, this is necessary for the cell that comprises is transported to and transported out of to nutrients and metabolin in described support. In the present invention, support also comprises or is made by following material, such as agar sugar, the poly-caprolactone (people such as Endres.M, Tissue Engineering, 2003, the 4th phase of the 9th volume, the 689-702 page or leaf), the coated carbon of niobium, shitosan, hydroxyl apatite-phosphoric acid three calcium (Harris, C.T. and Cooper, L.F., Comparison of matrices for hMSC delivery, 2004, the 747-755 page or leaf), collagen, hyalomitome acid, calcium phosphate, starch, hydroxyl apatite, fibrin, alginates, PVOH acid, carbon nano-fiber, polytetrafluoroethylene (PTFE), poly-lactic acid (Moran, the people such as J, Tissue Engineering, 2003, the 1st phase of the 9th volume, the 63-70 page or leaf) and composition thereof. Can also use among the present invention at United States Patent (USP) 6,231, the foam stand of describing in 879, it is based on the thermoplastic elastic body, for example polyamide, polyester, polyethylene, Kynoar, polyurethane (polyethyurethane) or polysiloxanes. In one embodiment, use the porous Merlon as timbering material.
The support that has wherein wrapped up the cell kind can have any rule or irregular (outside) shape. If support is for example to be used for skin tissue engineering, then the shape of support will be fit to the shape at the defective position that support will be used for. The support that uses in the present invention generally is that about 1 μ m is thick to about 5 μ m, can have in some embodiments the about 0.5cm of surface area2To about 20cm2。
In order to obtain the cell for skin equivalent of the present invention, the method that is used for isolation of stem/progenitor cells from amniotic membrane of umbilical cord has been described herein. Method comprises:
(a) in external other component separation with amnion and umbilical cord;
(b) under the condition that allows cell propagation, cultivate the amnion tissue that in step (a), obtains; With
(c) isolation of stem/progenitor cells.
For from the umbilical cord isolated cell, usually after (for the mankind's situation, baby) birth, collect immediately umbilical cord or its part, and in order to be transported to the laboratory, in the culture medium that is fit to the operation mammalian tissues, shift. The example of this type of culture medium includes but not limited to the Leibovitz culture medium, its can be in supplier such as Sigma-Alder gram company (Sigma Aldrich), Saint Louis, Missouri USA or U.S. Hyclone company (Hyclone), Logan, Utah, USA is purchased. Then, usually under aseptic condition, process umbilical cord. The processing of umbilical cord generally includes, and by the salt solution washing with suitable buffer solution such as phosphoric acid buffer, removes and remains in surface or the blood in the umbilical cord blood vessel. Then, usually umbilical cord is divided into less sheet, cuts as passing through, and again washing before separating amnion and other composition. In this, note it not being the umbilical cord that after birth, to process immediately the mammal donor, but may collect umbilical cord and optional after washing under the aseptic condition and being divided into more small pieces, preserve umbilical cord or its part by profound hypothermia-preservation, and the sample that will so obtain for example is stored in the liquid nitrogen, is used for later on from the umbilical cord isolated cell.
Term " profound hypothermia-preservation " used herein be its conventional sense, such processing is described, wherein by being cooled to low subzero temperature, for example (usually)-80 ℃ or-196 ℃ (boiling point of liquid nitrogen) are preserved cell or whole tissue. Can carry out profound hypothermia-preservation according to well known by persons skilled in the art, and can comprise the freezing-protective agent of use for example methyl-sulfoxide (DMSO) or glycerine, the formation of its umbilical cord intracellular ice crystal that slows down.
Refer to any cell of deriving from umbilical cord at term used herein " ancestral cells ", it has the ability that unlimited oneself upgrades and be divided into various kinds of cell or types of organization's (chrotoplast, upper chrotoplast, fibroblast, myocyte or neuron for example). Be not to need each experimenter of skin equivalent can provide umbilical cord as from the source of body ancestral cells (that is, from the cell of the umbilical cord amnion acquisition of the individual identical individuality that uses after a while skin equivalent of the present invention). Therefore, this paper also comprise xenogenesis (that is, in situation of the present invention, the umbilical cord amnion isolation of stem/progenitor cells of the species beyond the mankind) or allogeneic (namely, in situation of the present invention, from other people umbilical cord amnion isolation of stem/progenitor cells) purposes of ancestral cells. In addition, the cell that is used for the skin equivalent, and according to its production method of the present invention, can be from any mammalian species, for example mouse, rat, cavy, rabbit, goat, dog, cat, sheep, monkey or people, the cell in people source is preferred in one embodiment.
Term " embryonic stem cell sample characteristic " refers to the ability of the cell that umbilical cord is derived, namely they can---with embryonic stem cell almost or fully the same---spontaneously be divided into all types of organizations, mean that they are all can stem cell.
Term used herein " amnion " refers to wrap up the inner layer film capsule of developmental mammal embryo. Between the gestational period, fetus is surrounded and buffering by the liquid that is called amniotic fluid. This kind liquid is wrapped in the capsule that is called amnion together with embryo and placenta, and this capsule also covers umbilical cord. Owing to following reason amniotic fluid is most important. Amniotic fluid buffering and protection embryo allow the embryo to move freely. Amniotic fluid also allows umbilical cord floating, prevents that it is squeezed and prevents from cutting off the oxygen that derives from blood circulation in the placenta blood vessel and nutrient to embryo's supply. Amnion comprises the amniotic fluid of keeping Stable State Environment, and protection embryo environment is not subjected to external environment influence. This kind barrier also protects the embryo not to be subjected to also may to cause along row on the vagina impact of the biology (such as bacterium or virus) of infection.
Be used for culture medium that tissue cultivates and reagent well-known in this area (reference example as, Pollard, J.W. and Walker, J.M. (1997) Basic Cell Culture Protocols, second edition, Humana Press, Totowa, NJ; Freshney, R.I. (2000) Culture of Animal Cells, the 4th edition, Wiley-Liss, Hoboken, NJ). The suitable culture medium example that is used for cultivation/transhipment umbilical cord tissue sample includes but not limited to salt solution (PBS) and the L-15 culture medium of Dulbecco ' s improvement Eagle culture medium (DMEM), RPMI culture medium, CMRL 1066, Hanks ' balanced salt solution (HBSS), phosphoric acid buffer. Include but not limited to Dulbecco ' s improvement Eagle culture medium (DMEM), DMEM-F12, RPMI culture medium, CMRL 1066, EpiLife for the appropriate media example of cultivating ancestral cells of the present invention
Figure A20068004753500271
Culture medium and culture medium 171. Culture medium can be added the little cow's serum of tire (FCS) or tire cow's serum (FBS) and antibiotic, the growth factor, amino acid, inhibition thing etc., and these are entirely in technical staff's the general knowledge scope.
The method of isolation of stem/progenitor cells also comprises:
(a ") before cultivation, by enzymatic digestion and/or directly organize the outer planting body technique, in amnion tissue, separate these ancestral cells. Term used herein " enzymatic digestion technology " mean to add enzyme with cell from mainly organizing piece (referring to the umbilical cord amnion here) to discharge. Collect subsequently the cell that separates. Term used herein " is directly organized the outer planting body technique " and is meant at first tissue to be placed without in the enzyme culture medium. Subsequently, cell is gathered in the crops subsequently cell and is used for collecting voluntarily from mainly organizing piece to separate under meticulous condition.
Process or the method for directly organizing outer planting body method to separate the cell of specific tissue or organ is (reference example such as Pollard generally known in the art by enzyme, J.W. and Walker, J.M. (1997) Basic Cell Culture Protocols, second edition, Humana Press, Totowa, NJ; Freshney, R.I. (2000) Culture of Animal Cells, the 4th edition, Wiley-Liss, Hoboken, NJ). Any enzyme that the catalysis tissue can be dissociated is used for implementing the method. In an example, purpose is used the collagen enzyme for this reason. Use is as the enzyme of semifinished product or purified form. Described enzyme can produce from any biological (most preferably clostridium histolyticum (Clostridium the histolyticum)) purifying of prokaryotes or eucaryon or by the gene technology restructuring. Can use any type collagen enzyme, i.e. I type, II type, III type, IV type or its any combination. In some instances, preferably use the type i collagen enzyme.
In an example, the invention provides method for separating of the ancestral cells with embryonic stem cell sample characteristic. These cells finally can be divided on form, but are not limited to, epithelial stem cell or a mesenchymal stem cells.
Therefore, in another embodiment, the invention provides the skin equivalent, the cell of wherein deriving from ancestral cells is a mesenchymal stem cells (UCMC) or epithelial stem cell (UCEC). These cells (UCMC and UCEC) are that the method for separating epithelium and/or a mesenchymal ancestral cells obtains, and wherein disclose unanimously with above-mentioned, and these cells can have embryonic stem cell sample characteristic.
Epithelium ancestral cells (UCEC) comprises the arbitrary cell that shows chrotoplast sample form (being polygon), and it can be divided on any type chrotoplast for example chrotoplast, esophageal epithelial cell, intestinal epithelial cell, large intestine epithelium cell, lung and airway epithelial cell, Urothelial Cell or uterine epithelial cell on chrotoplast, liver epithelial cell, renal epithelial cell, the pancreas on chrotoplast, hair follicle cell, corneal epithelial cell, conjunctival epithelial cell, the retina on (but being not limited to) skin.
Between mesenchymal ancestral cells (UCMC) comprise the arbitrary cell of mesenchymal cell sample form between demonstration (being spindle sample shape), its can be divided into any type between the mesenchymal cell for example (but being not limited to) skin fibroblast, cartilage cell, Gegenbaur's cell, Tenocyte cell, ligament fibroblast, myocardium cell, smooth muscle cell, Skeletal Muscle Cell, fatty cell, derived from the cell of incretory and whole variants and the derived cell of neural ectoderm cell.
The method of isolation of stem/progenitor cells can also comprise:
(d) under the condition that allows cell generation clone to expand, cultivate ancestral cells.
Term " clone expands " (sometimes being also referred to as " mitosis sex clone expansion ") relates to the process of Development in the cell differentiation program, makes ancestral cells become particular lineage and eventually end differentiation of subsequently generation by this process. The condition that this area induces ancestral's cell clone to expand as everyone knows changes between different cell types obviously. Be not limited to specific method, induce the clone to expand usually and realize by optimizing for the grown cultures base cultivation ancestral cells of cell propagation. This type of culture medium multi-provider place that can comform obtains. The limiting examples of this type of culture medium is KGM
Figure A20068004753500291
-cutin forms cell culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), (the Kang Baisi company (Cambrex Corporation) of chrotoplast culture medium on the MEGM-breast, New Jersey, USA), EpiLife
Figure A20068004753500292
Culture medium (the Oregon of Ka Sikade biotech firm (Cascade Biologies Inc.), USA), Green ' s culture medium, the CMRL 1066 ((Mediatech of Medi-Tech. Inc, Inc.), Virginia, USA) or culture medium 171 (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA). Usually, these culture mediums need to add induce cell propagation reagent such as the growth factor. This type of reagent can be mixed in the single solution, forms the cell growth such as people's cutin and replenishes reagent box (Ka Sikade biotech firm (Caseade Biologies.), Oregon, USA) (only giving an example), perhaps can add individually. This type of reagent includes but not limited to grow, and (for example EGF, insulin-like growth factor-i, PDGF-BB, transforming growth factor-beta, cutin form cell growth factor (KGF to the factor; Be also referred to as HBGF-7 or FGF-7), TGF-α, two accent albumen), hormone (extracting thing such as Niu Chuiti), hydrogenation cortisone, transferrins and other any appropriate combination etc. expand with the clone who induces particular cell types. Term " clone expand " comprises that also cells in vivo cultivates, for example by with injection cell to mammal such as people, mouse, rat, monkey, ape.
The invention provides the skin equivalent, the natural composition that it has simulated skin has epidermal area and corium layer or has arbitrary layer in these two layers of skin layers. For this purpose, cell of the present invention UCMC and UCEC can be divided into respectively fibroblast and cutin forms cell for example. Therefore, the present invention provides the method for producing the skin equivalent in one embodiment, comprising:
● support is provided,
● will be placed in the described support from the cell that the ancestral cells that the umbilical cord amnion separates is derived or on the support, and
● hatch described support in the first culture medium, it allows described cell propagation and further differentiation.
As mentioned above, because the ancestral cells that separates from the umbilical cord amnion of the present invention has the potential that is divided into UCMC and UCEC, in some embodiments, in the method for producing the skin equivalent, preferred UCMC and the UCEC that derives from the ancestral cells of umbilical cord amnion separation that use.
In one embodiment of the invention, the UCMC extraction thing that obtains by description among the embodiment 20 can be used for inducing the cell from cell system to grow, and described cell is because their exact age does not breed fully under the ordinary student elongate member or not too breeds. Example as described in this article UCMC extraction thing can be used for inducing the growth that forms cell (asK) from the old and feeble cutin of patient's acquisition in 60 years old. Dermal fibroblast (NF) by using UCMC to extract other cell system that thing can induced growth, as describing among the embodiment 20.
Therefore, the present invention relates to induce the method for old and feeble keratinocyte propagation, be included in the suitable growth medium and cultivate old and feeble keratinocyte, and add the mescenchymal stem cell (UCMC) of umbilical cord amniotic membrane, to induce old and feeble keratinocyte propagation to the keratinocyte of aging.Method also comprise propagation old and feeble keratinocyte separation and be applied to them in the support or on the support.In one embodiment, can in 30 years old or above, 35 years old or above, 40 years old or above, 50 years old or above, 60 years old or above, 70 years old or above even experimenter more than 80 years old, separate old and feeble keratinocyte.Yet, can also from the experimenter younger, separate old and feeble keratinocyte than 30 years old.
The order of severity according to cutaneous wound or disease, it is just enough only to replace one deck, epidermal area promptly only is provided, skin corium perhaps only is provided, on described skin corium, place the keratinocyte of formation epidermis cultured or that obtain from other sources.In order to obtain epidermal area, UCEC can be divided into keratinocyte, and in order to obtain skin corium, UCMC can be divided into fibroblast.
Therefore, in the method for the invention, when described support comprised UCEC, first culture medium comprised and is suitable for the culture medium that keratinocyte is cultivated, thus propagation with break up described UCEC and become keratinocyte.
In this case, this first culture medium comprises keratinocyte growth medium, somatomedin, insulin, transferrins and Monohydrated selenium dioxide.
Somatomedin can be, for example epidermal growth factor (EGF), insulin-like growth factor-i, platelet derived growth factor-BB (PDGFb), transforming growth factor-beta, keratinocyte growth factor (KGF), TGF-α or amphiregulin.
The keratinocyte growth medium can be KGM for example
Figure A20068004753500311
-keratinocyte culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), EpiLife
Figure A20068004753500312
Culture medium (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA), Green ' s culture medium, the CMRL1066 ((Mediatech of Medi-Tech. Inc, Inc), Virginia, USA), M171 (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.
In one embodiment, when described support comprises UCEC, in order to breed and to break up described UCEC is keratinocyte, and first culture medium that is suitable for the keratinocyte cultivation comprises keratinocyte growth medium, epidermal growth factor (EGF), insulin, transferrins and Monohydrated selenium dioxide.In another embodiment, this first culture medium comprises EpiLife
Figure A20068004753500313
Culture medium (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA), epidermal growth factor (EGF), insulin, transferrins and Monohydrated selenium dioxide.In another embodiment, this first culture medium comprises about 98.8 to about 99.4% (v/v) EpiLife
Figure A20068004753500314
Culture medium (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and 10ng/ml epidermal growth factor (EGF).Can in Figure 18 a, see the example of the keratinocyte that the method for the application of the invention obtains.
In another embodiment of the inventive method, when described support comprises UCMC, be fibroblast in order to breed and to break up described UCMC, described first culture medium comprises the culture medium that is suitable for the fibroblast cultivation.In the case, first culture medium comprises fibroblastic growth culture medium and tire calf serum (FCS) or hyclone (FBS) simultaneously, is fibroblast thereby breed and break up described UCMC.The fibroblastic growth culture medium can be KGM -keratinocyte culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), EpiLife
Figure A20068004753500316
Culture medium (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), Green ' s culture medium, CMRL1066 (Mediatech company, Virginia, USA), M171 (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.In one embodiment; when described support comprises UCMC; in order to breed and to break up described UCMC is fibroblast, be used for first culture medium that fibroblast cultivates comprise about 90 to about 95% (v/v) fibroblastic growth culture medium and about 5 to about 10% tire cattle or tire calf serum (FCS).In another embodiment, first culture medium comprises about 90 to about 95% (v/v) CMRL1066 (Medi-Tech. Inc (Mediatech.Inc), Virginia is USA) with about 5 to about 10% tire calf serum (FCS).Can in Figure 18 b, see fibroblastic example of the method acquisition of the application of the invention.
In case UCMC or UCEC are divided into fibroblast and keratinocyte respectively fully, then can be used for the affected part of mammal or human body.But not only skin corium or epidermal area require displacement sometimes, but two-layer skin layer all requires displacement.This can be following situation, for example when the epidermal area of skin and skin corium because burn (holostrome wound) when destroying.Reach other purpose for this purpose, the invention provides method, it comprises
● support is provided,
● be placed on UCMC in the described support or on the support,
● hatch described support in first culture medium that is suitable for the fibroblast cultivation, it allows described UCMC propagation and further differentiation,
● be placed on UCEC in the described support or on the support and
● hatch described support in second culture medium, it allows described UCEC propagation and further is divided into keratinocyte.
Use is suitable for first culture medium that fibroblast cultivates (it comprise above-mentioned be divided into the identical component of fibroblastic first culture medium to being used for UCMC), those skilled in the art can be grown in support or on support comprise fibroblastic skin corium.Afterwards, can or use UCEC on the support in comprising this skin corium support.Use second culture medium (it comprises above-mentioned to being used for the identical component of first culture medium that UCEC is divided into keratinocyte), this epidermis skin layer of can on first skin corium, growing.Therefore, skin equivalent of the present invention can provide real, morphogenetic, multilamellar skin equivalent, it comprises deutero-fibroblast of UCMC-and the deutero-keratinocyte of UCEC-.Can see that in accompanying drawing 19 these skin equivalents provide the holostrome corium to regenerate, produce and accelerate healing and reduce scar.
Grow functional skin corium and need about 4 to 7 days time from being divided into fibroblastic UCMC, and in case grow skin corium and UCEC is incorporated in the support, also need other 8 to 10 days up to forming epidermal area from the deutero-keratinocyte of UCEC.For produce according to this area present situation from body cultured skin equivalent, according to bioptic size, need at least 21 to 35 days, yet use method of the present invention, only need 12 to 18 days.Except other reason, this also is due to the fact that, promptly uses the method for the present invention can accelerator, because cell bank of the present invention or existing cell culture provide the cell that is used for skin equivalent of the present invention (xenogenesis or allogeneic).The initial concentration that is used for UCMC and UCEC is about 1 * 10 in exemplary 5To about 1 * 10 6In cell/ml scope.In one embodiment, being used for inoculating UCMC is about 5 * 10 to the concentration of support 5UCMC/ml, what be used to inoculate UCEC is 1 * 10 6UCEC/ml.
As describing in background parts, the corium of natural skin not only contains fibroblast and also contains teleneuron, body of gland, hair follicle and blood vessel.In order further to improve the function of skin equivalent, therefore an embodiment of the inventive method comprises also that the cell with one or more cell lines is placed in the support or on the support, this cell line can be divided into blood vessel or body of gland.Some bodies of gland produce antiperspirant (sweat gland) to thermal response, and other body of gland produces oil (sebaceous gland) thereby maintenance skin wet softness.This oil is also as the barrier of resisting foreign substance.The blood vessel of corium provides nutrient for skin, and helps to regulate organism temperature.Because these extra cells are finished the critical function in the skin, one embodiment of the invention also provide method, only enumerate several examples, and its medium-height trestle also comprises vascular endothelial cell or microvascular dermal endothelial cell.In one embodiment, vascular endothelial cell is from mammiferous umbilical cord, in one embodiment, and from the mankind's umbilical cord.The limiting examples that can be used for the different cell lines of the inventive method is for example, only to enumerate several examples, human umbilical blood vessels endotheliocyte (HUVEC) or microvascular dermal endothelial cell (DMEC is called DMVEC again).
In addition, research shows that the function effect of cell extracellular matrix (ECM) pair cell is very big.As support, extracellular matrix forms tridimensional model, and its sustenticular cell growth also improves their function.Different with above-mentioned support, the natural material that extracellular matrix is produced by cell self constitutes, yet above-mentioned support can also comprise or be made up of artificial material, such as but not limited to the porous Merlon.Therefore, because the importance of substrate cell growth, reproducing ECM structure (centering on this substrate, the particularly substrate of analogue body inner tissue of cell in its better analogue body) is the main target of organizational project.Wei Tan, M.S. showed (Tissue Engineering with T.A.Desai, 2003, the 9th the 2nd phase of volume, 255-267 page or leaf) mixture of natural collagen, collagen and chitosan or the mixture of collagen, chitosan and fibronectin can be used for producing the substrate that is used for embedding therein human lung fibroblast and human umbilical vein endothelial cell.
Therefore, the present invention also provides method, wherein places at least a extracellular matrix component in described support or on the support.This extracellular matrix component should be simulated by the conventional ECM substrate that produces of cell self.Therefore, the ECM component is made up of the material that can also find in nature, produced by cell self.Preferably, should at least a extracellular matrix component with being placed in the support from the deutero-cell of above-mentioned ancestral cells or on the support.If be used for skin equivalent of the present invention can self producing the ECM component, then do not need manually to mix extracellular matrix component with the cell method of producing them.In one embodiment, after the ascorbic acid stimulation, UCMC oneself deposition collagen is as the ECM material.If also add the ECM component except the extracellular that the present invention uses, extracellular matrix component can be selected from material for example collagen, elastin laminin, intercellular adhesion molecule, laminin, heparin, fibronectin, Dan Baijutang, tenascin, FBN1 and composition thereof.If use collagen preferably only uses type i collagen in some embodiments at present or unites use I type and III Collagen Type VI.In one embodiment of the invention, use type i collagen.
Also described method, it further is included in them and is used for before the skin equivalent of the present invention the ancestral cells of isolating ancestral cells of preservation or differentiation (for example, UCMC and UCEC).
Be used to preserve and preserve eukaryotic cell especially the method for mammalian cell and scheme in this area.Interior well-known (reference example such as Pollard, J.W. and Walker, J.M. (1997) Basic Cell CultureProtocols, second edition, Humana Press, Totowa, NJ; Freshney, R.I. (2000) Culture of Animal Cells, the 4th edition, Wiley-Liss, Hoboken, NJ).Be used to keep isolating doing/group cell for example any means of epithelium ancestral cells or mesenchyme ancestral cells biologic activity can use jointly with the present invention.In an example, ancestral cells is kept and is preserved by using cryopreservation.
Therefore, also described by above method, and differentiation is from the cell of ancestral/stem cell from the deutero-ancestral/stem cell of umbilical cord amniotic membrane.In addition, also described cell bank, it comprises one or more isolating as mentioned above ancestral/stem cell or is made up of it.For example, the cell bank of this ancestral/stem cell can be to individuality from (for example, the latter is used for allograft subsequently) body or the property compiled, and can be applied to for example regenerative medicine, tissue repair and regeneration by further differentiation subsequently.
With above consistent, also can be included in the pharmaceutical composition from the isolating ancestral cells of umbilical cord amniotic membrane by said method.Pharmaceutical composition can also comprise the cell from the ancestral cells differentiation.Pharmaceutical composition can be an any kind, comprises ancestral cells usually, and the cell of its differentiation or its emiocytosis thing or cell extract have suitable pharmaceutically suitable carrier and excipient simultaneously.Under the situation of emiocytosis, can use the chemical compound that needs with the form of supernatant, wherein said chemical compound is secreted in the described supernatant.In another example, can handle supernatant, for example in being included pharmaceutical composition before purification or concentrate.In some instances, pharmaceutical composition is suitable for whole body application or topical application.
Being suitable for pharmaceutical composition for topical application can be liquid or sticky form.The example comprises ointment, ointment and lotion etc.The example that is suitable for the pharmaceutical composition that whole body uses is a fluid composition, and wherein ancestral cells or cell extract for example are dissolved in injectable or infusion with in the buffer.This class preparation of drug combination is those skilled in the art's the ken, and be described in for example Gennaro, A.L. and Gennaro, A.R. (2000) Remington:The Science and Practice ofPharmacy, the 20th edition, Lippincott Williams ﹠amp; Wilkins, Philadelphia is among the PA.
Therefore, the method for the treatment of the experimenter who suffers from disease has been described.This method comprises isolating as mentioned above ancestral cells from effective dose to the experimenter or the cell-derived since then cell extract of using.
In principle, can treat any disease or disease that is suitable for by the treatment of stem/progenitor cells method with cell or cell extract.Can also be the cell type that needs with cell differentiation, such as but not limited to, Skin Cell, bone or chondrocyte, hepatocyte, antigen-production cell, hormone are produced for example β islets of langerhans insulin production cell of cell, and the cell of curative use differentiation.
Therefore, the invention still further relates to the method for generation insulin-production cell, comprise
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane and
● in proper culture medium propagation with break up described cell and become β-islet cells.
In one embodiment, be mescenchymal stem cell (UCMC) or epithelial stem cell (UCEC) from the deutero-cell of ancestral cells, by comprise nicotiamide in cell culture medium, it can further be divided into the β-islet cells of excreting insulin.
Except nicotiamide, culture medium can also comprise the growth medium that is used for β-islet cells cultivation.Culture medium can also comprise that somatomedin or fetal blood are clear.Fetal blood can be clearly for example calf or Niu Laiyuan.In one embodiment, culture medium can also comprise insulin, transferrins and Monohydrated selenium dioxide.Somatomedin can be such as but not limited to epidermal growth factor (EGF), insulin-like growth factor-i, platelet derived growth factor-BB (PDGFb), transforming growth factor-beta, keratinocyte growth factor (KGF), TGF-α or amphiregulin.The growth medium that is used for β-islet cells cultivation can be such as but not limited to KGM
Figure A20068004753500361
-keratinocyte culture medium (Kang Baisi company (CambrexCorporation), New Jersey, USA), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), EpiLife
Figure A20068004753500362
Culture medium (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), Green ' s culture medium, CMRL1066 (Medi-Tech. Inc (Mediatech.Inc), Virginia, USA), M171 (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.
In one embodiment of the invention, use the culture medium differentiation UCMC or the UCEC that in embodiment 15, describe to become β-islet cells.
In another embodiment, method comprises separates the insulin that β-islet cells is produced, and it can be used for the treatment of for example insulin-dependent diabetes (IDDM) then, as in mammal.Mammal can be for example mankind, cat, Canis familiaris L., sheep, horse or pig.Can be according to such as but not limited to JonesP.M. and Saermark, people such as T (Anal Biochem.1987Oct; 166 (1): 142-9) method of Miao Shuing is carried out the separation of insulin.
Therefore, the invention still further relates to the insulin-production cell of the method acquisition that produces the insulin production cell according to the present invention.The invention still further relates to the method for treatment and the unbalance relevant disease of insulin level, comprise to the insulin-production cell of administration by the inventive method acquisition.Mammal can for example be people, cat, Canis familiaris L., sheep, horse or pig.The example of this type of disease is insulin-dependent diabetes (IDDM).
In another embodiment of the invention, the method that produces dopamine and tyrosine hydroxylase production cell is provided, comprise that cultivation is from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane, preferred UCMC and in proper culture medium with described cell proliferation be divided into dopamine and tyrosine hydroxylase (TH) is produced cell.Dopamine plays a role as neurotransmitter, activates dopamine receptor.Dopamine still is the neuro hormone that hypothalamus discharges.It is to suppress antepituitary to discharge prolactin antagonist as the major function of hormone.Dopamine can be used as medicine and provides, and acts on sympathetic nervous system, produces the effect of for example heart rate quickening and hypertension.Tyrosine hydroxylase is to be used for the responsible catalytic amino acid of epinephrine building-up process-L-tyrosine at body to be converted into the dopamine precursor---the enzyme of dihydroxyphenylalanine (DOPA).Therefore, in another embodiment, method also comprises dopamine and/or the tyrosine hydroxylase of separation from deutero-dopamine of UCMC and the generation of tyrosine hydroxylase production cell.
The present invention also provides the method that produces human leucocyte antigen (HLA) G (HLA-G).Human leucocyte antigen (HLA) (HLA)-G is a major histocompatibility complex I class antigen, and it is called as non-classical, because it has showed the distribution of tissue limitations, the cytoplasm domain of minimizing, limited polymorphism and some isotypes in Placenta Hominis.Think that HLA-G has played important function at pregnancy duration, by protecting semiallogenic fetus not by maternal immunity cell recognition and destruction.Relate to HLA-G in the disease of panimmunity mediation and the disease, as organ-, cell transplantation and autoimmune disease.The example of this type of autoimmune disease is multiple sclerosis, rheumatic arthritis, type i diabetes, psoriasis, thyroid disease, systemic lupus erythematosus (sle), scleroderma or celiac disease.Therefore, the invention provides and produce the method that human leucocyte antigen (HLA) G (HLA-G) produces cell, comprise cultivation from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane, with propagation in proper culture medium with break up described cell and become HLA-G to produce cell.Can produce HLA-G production cell from UCMC or UCEC.Use method of the present invention, show for the first time inmature UCEC (
Figure A20068004753500371
UCEC) express and produce HLA-G.Surprising, for this method, the specific UCEC that induces produces HLA-G not necessarily (referring to embodiment 19).
Other can be selected from oncosis, accelerating type skin aging and dermatosis, tissue disorder, internal organs endocrine defects (visceral endocrinedeficiency) and neurological disorder with the disease of the ancestral cells treatment of describing herein.
Tissue disorder to be treated can be congenital or acquired tissue defects.Can include but not limited to testosterone defective, anemia, hypoglycemia, hyperglycemia, pancreas defective, adrenal gland's defective and thyroid defective with ancestral cells or from the example of the internal organs endocrine defects of its deutero-cell therapy.
The example of treatable neurological disorder includes but not limited to, A Zihai Mo's disease, parkinson disease, Jacob Kreutzfeld ' s disease, Lou Gehrig ' s disease, Huntington Chorea and neural tumor disease.
The invention still further relates to the purposes from the isolating mescenchymal stem cell of umbilical cord amniotic membrane (UCMC), it is used to produce osteocyte (referring to embodiment 10) (being used for the treatment of bone injury), or produces chondrocyte (referring to embodiment 17) (being used for cartilage injury's treatment).
In addition, the present invention also provides the hepatocellular method that produces, and comprises cultivation from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane, preferred UCEC, with propagation in proper culture medium with break up described cell and become hepatocyte.Proper culture medium comprises Oncostatin .-M, and it is used to be induced to differentiate into hepatocyte.Oncostatin .-M (OSM) is polytropism cell pyrimidine (pleitropic cytocine), belongs to the interleukin-6 group of cytokine.In these cytokines, it is all the most similar on 26S Proteasome Structure and Function to leukaemia inhibitory factor.Yet, even it is not accurately defined and is proved to be in liver growth, hemopoiesis, inflammation and may is important in CNS grows.
Consistent with above-mentioned discussion, the invention still further relates to the method for the treatment of wound or dermatosis, comprise wound or dermatosis are used skin equivalent of the present invention.Therefore, the skin equivalent of the present invention that obtains by method of the present invention can be used for the production of pharmaceutical composition.The pharmaceutical composition of treatment burned skin, ulcer, radiation and the diabetic wound that the invention still further relates to so obtain.The invention still further relates to the cell bank that comprises skin equivalent of the present invention.
Dermopathic example is the wound or the damaged portion of part skin, for example, and the skin of sun burns.In this article, the aging of skin also is considered to dermatosis.Ancestral cells or its cell extract local or similarly send, for example, as the component in lotion or ointment or any other suitable carrier, can therefore be used to repair the skin of sun damage, can slow down the in addition process (defying age character) of skin aging, thus this is to strengthen the somatomedin and relevant peptide element (lack them and will accelerate skin aging) that lacks by replenishing.Therefore can use skin equivalent of the present invention.The affected area that ancestral cells can also be moved to body is surface injury for example, forms the necessary elemental cell of local repair process (referring to The Journal of Immunology, 2001,166:7556-7562; Or InternationalJournal of Biochemical and Cell Biology 2004; 36:598-606).
Tumor disease can be a cancer, and is special because research has recently confirmed optionally target tumor tumor tissue (Journal of the National Cancer Institute 2004 of stem cell; 96 (21): 1593-1603), allow antitumor agent such as interferon orientation sent and pass to the tumorigenesis kitchen range.Cancer can be the cancer of any kind, comprises those cancers that can form solid tumor, scope from skin carcinoma to visceral cancer.The example of cancer to be treated comprises squamous cell carcinoma, breast duct and lobular carcinoma, hepatocarcinoma, nasopharyngeal carcinoma, pulmonary carcinoma, osteocarcinoma, cancer of pancreas, skin carcinoma, head or cervical region cancer, skin or ophthalmic malignant melanoma, uterus carcinoma, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast carcinoma, carcinoma of testis, uterus carcinoma, carcinoma of fallopian tube, carcinoma of endometrium, cervical cancer, cancer of vagina, carcinoma vulvae, Hodgkin, non-Hodgkin lymphoma, esophageal carcinoma, carcinoma of small intestine, the hormonal system cancer, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, carcinoma of urethra, carcinoma of penis, carcinoma of prostate, chronic or acute leukemia, child's solid tumor, lymphocytic lymphoma, bladder cancer, kidney or carcinoma of ureter, renal cell carcinoma, carcinoma of renal pelvis, central nervous system (CNS) tumor, constitutional CNS lymphoma, tumor-blood-vessel growth, the spinal column axis tumor, the brain stem glioma, pituitary adenoma, Kaposi sarcoma, the combination in any of epidermoid carcinoma or this carcinoid comprises its distribution (transfer) form.When the treatment tumor disease, deutero-stem cell of umbilical cord amniotic membrane described herein and/or their cell extract both can be used as direct treatment and/or had used as carrying the carrier general.When antineoplaston, cell comprises anti-tumor agents.
In another pharmaceutical use, ancestral cells can be used for gene therapy.For this purpose, cell can be transformed with being coded in proteinic nucleic acid to be produced in the cell.Any means in can the well-known several different methods of operation technique personnel imports cell of the present invention with nucleic acid, for example use viral vector and/or contain the transfection group compound of lipid such as IBAfect (IBA GmbH,
Figure A20068004753500391
Germany), Fugene (F. Huffman-La Luoqie company (F. Hoffmann-LaRoche Ltd.), Basel, Switzerland), Gene Porter (gene therapy system house (Gene Therapy Systems)), Lipofectamine (hero Life Technologies, Inc. (Invitrogen Corporation), California, USA), Superfect (fast and smart company (Qiagen), Hilden, Germany), Metafecten (rich Plutarch (the Biontex of company difficult to understand, Munich, Germany) or at PCT application WO 01/015755) in those transfection group compounds of description.In relevant embodiment, ancestral cells and derived from its cell after the nucleic acid with the selected polypeptide of coding transforms, can be used for reorganization and produce this polypeptide.
As mentioned above, the stem cell extract is rich in multiple somatomedin and the peptide relevant with normal structure physiology.This type of somatomedin and/or peptide may lack in the body exposed portions, as not influenced with in the skin of keeping inner stable state by extraneous factor as proprietary top layer protection body.Therefore, ancestral cells or its cell extract are suitable for treatment and/or keep inner stable state.
In addition and with as above disclose consistently, can be used to produce any biomolecule with ancestral cells or derived from its cell.Biomolecule can be any molecule of natural generation or its code nucleic acid molecule by the recombinant DNA technology transfered cell in cell for example.Can include but not limited to protein such as cytokine by the branch sub-instance that cell produces, somatomedin such as insulin like growth factor (IGF), epidermal growth factor (EGF), transforming growth factor (TGF β), activator protein A, bone morphogenetic protein (BMP), PDGF or hormone such as insulin or erythropoietin or transport protein such as transferrins, peptide such as somatomedin or hormone (Alfasone (LSH) for example, follicle stimulating hormone (FSH)), organic molecule such as steroid hormone, oligosaccharide or polysaccharide be heparin or Heparan sulfate (for embodiment in this respect with reference to WO 96/23003 or WO 96/02259) for example, Dan Baijutang, glycoprotein such as collagen or laminin or fat etc.
Mucoprotein, it is a glycoprotein, is the complicated molecule that can find in the viscous secretion (for example saliva, gastric juice, chyle, bronchus liquid) of most of epithelial layers.Lubricated and the defencive function of mucoprotein execution (for example, the transportation of chime, cushion excessive gastric acid, the lubricating function of knuckle synovia).Owing to its labyrinth and its high molecular weight (about 100 ten thousand-5,000 ten thousand dalton), be difficult to the mucoprotein molecule of separating natural form usually.
Therefore, the present invention also provides the method that produces mucoprotein-production cell, comprising:
● umbilical cord amniotic membrane epithelial stem cell (UCEC) is placed in the container (for example, culture bottle, culture dish) and
● in the culture medium that is fit to the secretory cell cultivation, hatch described UCEC.
These mucoprotein-production cells not only can be used for separating mucoprotein from cell culture medium, but also can use these cells in the method for the invention, comprise making the tissue that comprises the cell that is influenced by cigarette contact the mucoprotein-production cell that produces by the inventive method.These cells that are affected can be for example lungs of respiratory tract, or the cell on eye surface.
In addition, the mucoprotein-production cell that obtains by the inventive method can be used for treating synovial cell's sarcoma, cigarette sucks damage or eye surface damage.By the inventive method obtain mucoprotein-produce cell and also can be used for esophagus and air flue organizational project, be used for cosmetic applications or as genes matter delivery system.
For break up UCEC described herein become mucoprotein-produce cell, that invention also provides culture medium, wherein said culture medium to comprise to be used for is mucoprotein-produce growth medium, insulin, transferrins, Monohydrated selenium dioxide and the somatomedin of cell culture.
Somatomedin can be, such as but not limited to epidermal growth factor (EGF), insulin-like growth factor-i, platelet derived growth factor-BB (PDGFb), transforming growth factor-beta, keratinocyte growth factor (KGF), TGF-α or amphiregulin.
The growth medium that is used for mucoprotein-production cell culture can be, such as but not limited to KGM
Figure A20068004753500411
-keratinocyte culture medium (Kang Baisi company (Cambrex Corporation), NewJersey, USA), MEGM-breast epithelial cell culture medium (Kang Baisi company (CambrexCorporation), New Jersey, USA), EpiLife
Figure A20068004753500412
Culture medium (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), Green ' s culture medium, CMRL1066 (Medi-Tech. Inc (Mediatech.Inc), Virginia, USA), M171 (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), L-15 culture medium, Dulbecco ' s improve Eaglo culture medium (DMEM), DMEM-F12 and RPMI culture medium.
In one embodiment, be fit to the mucoprotein-culture medium of producing cell culture comprise be used for mucoprotein-produce growth medium, epidermal growth factor (EGF), insulin, transferrins and the Monohydrated selenium dioxide of cell culture.
In another embodiment, be fit to the mucoprotein-culture medium of producing cell culture comprise about 98.8 to about 99.4% (v/v) be used for mucoprotein-produce cell culture growth medium, about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and about 10ng/ml epidermal growth factor (EGF).
In another embodiment, the culture medium that is fit to mucoprotein-production cell culture comprises about 98.8 to about 99.4% (v/v) CMRL1066 (Medi-Tech. Inc (Mediatech.Inc), Virginia, USA) or M171 (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon, USA), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and about 10ng/ml epidermal growth factor (EGF).
Can define the mucoprotein-production cell that produces by method of the present invention with mucoprotein grumeleuse test (referring to embodiment 16).This test also is described in Corfield A.P., Glycoprotein method andprotocols:The Mucins, 29-65 page or leaf .Humana Press 2000; Gatter R.A. and Schumacher R.H., A practical handbook of join fluid analysis, 59-63 page or leaf, Lea﹠amp; Febiger, in 1991, its complete content is incorporated herein by reference.This test is to the mucoprotein quality of producing by the UCEC that cultivates in the cell culture medium of above-mentioned definition and the assessment of quantity.In brief, in the test media of this cell culture, inject the 7N glacial acetic acid, in described culture medium the UCEC cell the method according to this invention hatch.Acetic acid causes mucoprotein formation grumeleuse.Contain mucoprotein culture medium will show as have closely, the clear liquid of thickness grumeleuse.Therefore, in one embodiment, mucoprotein production cell used herein refers to, produces the cell of positive findings when detecting with test of describing among the embodiment 16 and condition.
Consistently with nearest method (see for example Amit, M etc., Human Feeder Layers for humanembryonic stell Cells, Biol Reprod 2003; 68:2150-2156), the ancestral cells of Miao Shuing can be used as the feeder layer of cultivating other embryonic stem cell, particularly human embryo stem cell herein.In one of these embodiments, cell is people source preferably, because end user's cell makes animal derived components as feeder layer, and for example animal pathogen or immunogen, the risk minimization of contamination of cells culture.Aspect this, should also be noted that, ancestral cells can be cultivated and from its deutero-cell under serum-free condition, therefore, use cell as feeder layer and under serum-free medium the cultured cell culture, described culture medium for example at this paper described below or in people such as Draper (Culture andcharacterization of human embryonic stem cell lines, Stem Cells Dev 2004 describes 13:325-336) or in International Patent Application WO 98/30679.
In this context, should be noted that a large amount of low generation cell (being the high-quality cell of vast scale) with minimum scale senile cell is vital and need derives in spare-part surgery and the therapy based on cell in the possible shortest time during cell expands.For example, from the mescenchymal stem cell amount of bone marrow and Cord blood few and therefore need the long period through numerous expansions of going down to posterity to reach the needed enough cell numbers of cell transplantation.Yet high generation cell is quality degradation and may cause cell ageing or carcinous conversion often.Have been found that herein and can use repeated explant technology to obtain a large amount of ancestral cells with less passage number.Therefore described the method for cultivating ancestral cells, wherein this method comprises:
Obtain organizing explant from the umbilical cord amniotic membrane;
In suitable culture medium and under the condition of culture this tissue explant is cultivated in the stage between when appropriate,
Randomly make and organize explant contact fresh culture and the suitable time phase of continuous culture (with reference to Figure 15) under appropraite condition.
In a single day cultivation can be implemented and obtain required cell number promptly to stop by circulation (going down to posterity) number of times as required.Make and organize explant contact fresh culture by removing exhausted cell culture medium from the container that is used for cell growth and fresh culture being added this container.Except to replacing in the used container the culture medium, can realize the contact fresh culture by the new container that will organize explant to be transferred to be full of culture medium.The explant of organizing that is used for cell culture and/or propagation can obtain by appropriate methodology, for example by aforesaid " directly organizing the explant technology " (at first tissue is placed therein in the no enzyme culture medium, cell separates and harvesting is used for collecting subsequently from main piece of tissue voluntarily under meticulous condition subsequently).
Organize the cultivation of explant in being applicable to any culture medium of cultivating mammalian cell, to implement.Just cultivate or the clone expands ancestral cells and derived from regard to its cell, the culture medium example comprise routine as listed above with the available culture medium of commerce, for example be not limited to KGM
Figure A20068004753500431
-keratinocyte culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA), EpiLife
Figure A20068004753500432
Culture medium (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA), culture medium 171 (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA), DMEM, DMEM-F12 or RPMI culture medium.Cultivate usually just being usually used in cultivating the derive condition (temperature, gas) of kind cell of this cell and implement down, for example in 37 ℃, 5%CO 2Air in.In one embodiment, use serum-free, particularly do not have the Ox blood serum culture medium and implement to cultivate.Usually, continue to cultivate (in a generation) to cell and grow the needed any appropriate time, continue for example about 7 days of about 1 to a couple of days or about 8 days but never be limited to.
Enforcement under the clear and definite disclosed arbitrary portion of this paper, the restriction can lacked in the illustrational the present invention of this paper.Therefore term " comprises ", " comprising ", " containing " wait and should do popularity and understanding without limitation.In addition; used term and statement herein used as descriptive and non-limiting term; and when using this class term and statement, do not get rid of the intention that institute shows and describe the equivalent of characteristic or its part, still confirm that multiple adjustment still may be in the scope of the present invention's claim required for protection.Therefore, should be understood that, though the present invention specifically discloses by embodiment preferred and optional characteristic, adjustment of the present invention and the change that the disclosure embodied be will be apparent to those skilled in the art, and this type of adjustment and change are considered as within the scope of the present invention.
In this article the present invention extensively also usually is described.The narrower kind and the subclass genus group that are in the open scope of generic attribute have also formed part of the present invention.This comprise of the present invention have since then remove the collateral condition of any theme in belonging to or the generic attribute of the present invention of negativity restriction is described, whether the material of no matter being got rid of is quoted in this article especially.
Other embodiment is in following claim and the non-limiting example scope.In addition, when term description is organized with Ma Kushi in feature of the present invention and aspect, those skilled in the art will recognize that therefore the present invention also describes according to any individual member or the subtribe member of this Ma Kushi group.
Embodiment
Embodiment 1: umbilical cord tissue is collected
Collect umbilical cord tissue behind the baby due immediately.Specimen drip washing is clean and be transferred to immediately before being transported to laboratory and contain culture transhipment culture medium and (add the L-15 culture medium of 50IU/ml penicillin, 50 μ g/ml streptomycins, 250 μ g/ml amphotericins, 50 μ g/ml gentamycins; All reagent are all available from Invitrogen) the aseptic vial of 500ml in.In laboratory, in the clean by laminar flow cabinet, extracting stem cell under the aseptic condition.At first specimen is transferred to aseptic rustless steel pallet.Use add the 5IU/ml heparin (from Sigma-Alder Riker Inc. (Sigma-Aldrich), Missouri, all remained blood in the cord vessels are removed in the washing of saline solution (PBS) multiple injection of warm phosphoric acid buffer USA).Use the common PBS of no heparin the last time in the washing.Subsequently the umbilical cord tissue specimen is cut into the small pieces of long 2cm and is transferred in the Tissue Culture Dish of diameter 10cm, with further washing and the sterilization of 70% ethanol, use subsequently and contain antibiotic cocktail (50IU/ml penicillin, 50 μ g/ml streptomycins, 250 μ g/ml amphotericins, 50 μ g/ml gentamycins therein; All available from hero Life Technologies, Inc. (Invitrogen Corporation), California, many washings of PBS USA) are clear until solution becomes.
Embodiment 2: cell separation/cultivation
At first block umbilical cord tissue so that umbilical cord amniotic membrane and jelly of Wharton (being umbilical cord matrix) and other internal component are separated.Subsequently isolating amniotic membrane is cut into that (0.5cm * 0.5cm) small pieces are used for cell separation.The umbilical cord membrane film is placed on the tissue culture's ware under the different cell culture conditions that are used to separate epithelial stem cell or mescenchymal stem cell and implements outer planting.
For mesenchymal cell separation/cultivation, explant is immersed in 5ml adds 10% hyclone (U.S. Hyclone company (Hyclone), Utah, USA) DMEM (hero Life Technologies, Inc. (Invitrogen Corporation), California is USA) in (DMEM/10%FBS) and at CO 2Cell culture incubator is interior in 37 ℃ of cultivations.Culture medium was changed once in per 2 days or 3 days.The cell outgrowth is checked under optical microscope.(0.125% trypsin/0.05%EDTA) results are used for further expanding and using culture medium DMEM/10%FBS cryopreservation to the cell that grows by trypsinization.
For epithelial cell separation/cultivation, before tissue sample being positioned over the surface with 1 Collagen Type VI/4 Collagen Type VI mixture (1: 2) coated cell culture dish frostings.Tissue sample is immersed in (both are all from the Oregon of Ka Sikade biotech firm (Cascade BiologiesInc.), USA) in 5ml EpiLife culture medium or the culture medium 171.Culture medium was changed once in per 2 days or 3 days.Under optical microscope, check cell growth from tissue culture's explant.The cell that grows is by trypsinization (0.125% trypsin/0.05%EDTA) use EpiLife culture medium or culture medium 171 to gather in the crops.
For the enzyme extraction method of cell, the umbilical cord amniotic membrane is divided into the small pieces of 0.5cm * 0.5cm and 37 ℃ of digestion 6 hours in 0.1% (w/v) type i collagen enzymatic solution (L. Huffman-La Luoqie company (L.Hoffmann-La RocheLtd.) Basel, Switzerland).With per 15 minutes vortex mixed of sample 2 minutes.4000 rev/mins of centrifugal 30 minutes collecting cells.Adopt two kinds of distinct methods so that separate epithelial stem cell or mescenchymal stem cell.
For the separation of epithelial stem cell, cell precipitation is resuspended in adds 50 μ g/ml insulin-like growth factor-is (IGF-1), 50 μ g/ml platelet derived growth factor-BB (PDGF-BB), 5 μ g/ml transforming growth factor-beta (TGF-β 1) and 5 μ g/ml insulins (all from R﹠amp; (the R﹠amp of d system company; DSystems), Minneapolis, USA obtains) the EpiLife culture medium or culture medium 171 (both are all from Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA) in, counting was also being used 1 Collagen Type VI/4 Collagen Type VI mixture (1: 2 in advance; Becton Dickinson and Co. (Becton Dickinson), New Jersey, USA) bag quilt 10cm tissue culture ware in 1 * 10 6The density inoculation of individual cell/ware.After 24 hours, the cell that adheres to is replaced former culture medium with warm PBS washing and with EpiLife culture medium of adding fill-in or culture medium 171.Culture medium was changed once in per 2 days or 3 days.Cell growth and the clone who expands are formed under the optical microscope and check.When about 70% converged, (0.125% trypsin/0.05%EDTA) inferior cultivation was used for further expanding and cryopreservation by trypsinization with cell.
For the separation of mescenchymal stem cell, with cell precipitation be resuspended in the PTT-4 culture medium, counting and in 10cm tissue culture ware with 1 * 10 6The density inoculation of individual cell/ware.Culture medium was changed once in per 2 days or 3 days.Cell growth and expansion are checked under optical microscope.When about 90% converges, with cell time cultivation as mentioned above.
For on feeder layer, cultivating epithelial stem cell and mescenchymal stem cell, the umbilical cord inner lining film is handled digestion, counted and be inoculated in the Green ' s culture medium in the 10cm tissue culture ware of 3T3 fibroblast (feeder layer) the bag quilt that lethal exposure or ametycin are handled by collagenase.Culture medium was changed once in per 2 days or 3 days.Colony is formed on to be checked under the optical microscope and photograph.
Embodiment 3: ancestral cells is identified
The epithelial cell image that the umbilical cord amniotic membrane that epithelial cell: Fig. 1 demonstration prepares from using-system explant method grows (40 * amplify).Image is in tissue culture the 2nd day (Figure 1A) and (Figure 1B C) took in the 5th day.The morphocytology analysis shows the epithelioid cell of polygon.Produce similar (Fig. 2) epithelial cell (40 * amplification) by the segmental enzymic digestion of umbilical cord at the 2nd day (figure A, C) and the 5th day (figure B, D).Fig. 7 shows from using Green ' s method epithelial stem cell colony of cultured umbilical amniotic membrane on feeder layer to form image (40 * amplification).The epithelioid cell colony of polygon enlarged rapidly on the from the 3rd to the 7th day.
Mesenchymal cell: place the back in as tissue culture's ware of culture medium at CMRL-1066 (or PTT-4 culture medium) that 10% tire calf serum (FCS) is added in use and early observed the mesenchymal cell outgrowth that grows out (Fig. 3 A, C) that shifts out from the umbilical cord amniotic membrane (40 * amplify) to 48 hours.Cell is characterised in that they are the spindle form and not only move easily but also promptly and expand external, with fibroblast very similar (Fig. 3 B, D) (40 * amplify).In by collagenase enzymic digestion isolated cells group, show similar observed result (Fig. 4).Fig. 4 A is presented at second day from the isolating mesenchymal cell of umbilical cord amniotic membrane.Observed cell proliferation (Fig. 4 B) (40 * amplification) at the 5th day.The colony from the mescenchymal stem cell of umbilical cord amniotic membrane that Fig. 6 and 8 is presented at no feeder layer (Fig. 6) and has under the feeder layer condition that (Fig. 8-1 uses the 3T3 feeder layer) cultivate in the PTT-4 culture medium forms image (40 * amplify).The colony of the fibroblast-like cell of elongated shape enlarged rapidly on the from the 3rd to the 7th day.In noticing in this regard, the 3T3 feeder layer suppresses the growth of mesenchymal cell usually, and is the same with human skin fibroblast.Again, the corresponding bulk phase ratio of this behavior that has shown mesenchymal cell of the present invention and more differentiation is different.
In other experiment, studied the clonality of the mesenchymal cell (UCMC) of invention.Form efficiency test for the clone, 100-200 of plantation is unicellular in the 100mm of no feeder layer tissue culture's ware or T75 bottle.Cell was kept in DMEM/10%FCS 12 days.The monitoring monoclonal forms (experiment of UCMC-16 and UCMC-17 in Fig. 8-2 is carried out in experiment in duplicate) under the optical microscope being inverted.Take microphotograph continuously.At the 12nd day, fixing colony and dye with rhodamine.Observe UCMC colony forming unit (Fig. 8-2).Observed a plurality of large-scale colony represents that CLSC is at external self renewal (Fig. 8-2).
Western engram analysis (Fig. 9) shows isolating mescenchymal stem cell from the umbilical cord amniotic membrane (UCMC) and umbilical cord epithelial cell (UCEC) expression coding embryonic stem cell specificity marker thing according to the present invention, the POU5f1 gene of transcription factor octamer-4 (Oct-4) is (with reference to Niwa, H., Miyazaki, J. and Smith, A.G. (2000) .Nat.Genet.24,372-376) (Fig. 9-1).Other result who shows among Fig. 9 .1a and the 9.1b confirms the expression of Oct-4 in the UCMC cell.In brief, in the experiment of example results, UCMC is planted in the 100mm tissue culture ware with the density of 50 cell/culture dishs in obtaining Fig. 9 .1a and 9.1b.Then, cell was kept 10 days in PTT-4 (composition of PTT-4 is referring to embodiment 12), as seen up to some clones.Then, fixing clone and with anti-Oct-4 antibody incubation (ES cell sign matter sample test kit (article No. SCR002); Triumphant Micon A.S. (Chemicon), Temacula, CA).A culture dish (No.5 among Fig. 9-1a) is as negative control, only with two anti-dyeing.Fig. 9 .1b is presented at the form of the painted UCMC cell of growing in the above-mentioned PTT-4 culture medium.Therefore, this analysis has shown embryo's sample characteristic of these stem cell.These mesenchymes and epithelial cell also are expressed as the mark Bmi-1 of soma cell self-renewal needs (referring to people such as Park, J.Clin.Invest.113,175-179 (2004) (Fig. 9-27), with the leukaemia inhibitory factor (LIF) (Fig. 9-28) that is considered to keep stem cell and blastocyte versatility, therefore, it is used to separate and the amplification human nerve stem cell.These cells are also highly expressed other somatomedin such as Connective Tissue Growth Factor (CTGF) (Fig. 9-6,9-7), VEGF (VEGF) (Fig. 9-10,9-11), Placenta Hominis like growth factor PLGF (Fig. 9-4,9-5), STAT3 (Fig. 9-2,9-3), stem cell factor (SCF) (Fig. 9-16), hepatocarcinoma derivative growth factor (HDGF) (Fig. 9-14,9-15), fibroblast growth factor-2 (FGF-2) (Fig. 9-12,9-13), platelet derived growth factor (PDGF) (Fig. 9-8,9-9), α-smooth muscle actin (α-SMA) (Fig. 9-17), fibronectin (Fig. 9-18,9-19), decorin gene polysaccharide (Fig. 9-20), cohere Dan Baijutang-1,2,3,4 (Fig. 9-21 is to 9-26).In Fig. 9, with mesenchymal cell (ADMC) comparison of these expression of gene and human dermis fibroblast, medulla mesenchyma cell (BMSC) and adipose-derived.Fig. 9-29 shows the Western trace data of UCEC and UCMC secretion leukaemia inhibitory factor (LIF).Fig. 9-30 shows, compare with epidermal keratinocytes with stem cell, the human dermis fibroblast of bone marrow, adipose-derived, in the supernatant of umbilical cord mesenchymal stem cells and epithelial stem cell culture, height secreted activator protein A and the Follistatin of measuring (Fig. 9-30) detection by ELISA are (well-known, these two kinds of protein all promote tissue repair and regeneration, enhancing blood vessel to take place and keep embryonic stem cell and cultivate, so each expression of gene is the sign of embryo's sample characteristic and cell differentiation ability).These results show that also cell of the present invention is the promising candidate target that these cell field treatments are used, described cell field such as regenerative medicine, old and feeble medical science, tissue repair and organizational project.In addition, the ability of Fig. 9-29 and 9-30 showed cell secreting, expressing product in culture medium.
By further characterizing mesenchymal cell with excretory cytokine of human marrow mesenchymal stem cell comparative analysis and somatomedin.With umbilical cord epithelial stem cell (UCEC) and human epidermic keratinocyte's comparative analysis.Followingly carry out this analysis: in brief, with UCMC, UCEC, dermal fibroblast, medulla mesenchyma cell, epidermal keratinocytes be incubated in the growth medium until 100% converge (37 ℃, 5%CO 2), subsequently hungry culture medium (serum-free DMEM) inter-syncization 48 hours.Change culture medium into fresh serum-free DMEM once more next day, subsequently cell cultivated 48 hours again.((RayBiotech, Inc), GA USA) analyzes in Lei Shi biotech company conditioned medium to be collected, concentrated and uses the cytokine array.
This analysis result shows, UCMC secreting leukocytes mesonium-6 (IL-6); (MCP1); Hepatocyte growth factor (HGF); Interleukin 8 (IL8); STNFR1; GRO; TIMP1; TIMP2; TRAILR3; UPAR; ICAM1; IGFBP3; IGFBP6 (Figure 11), and UCEC secretion IGFBP-4; PARC; EGF; IGFBP-2; IL-6; Angiogenin; GCP-2; IL1R α; MCP-1; RANTES; SCF; TNF β; HGF; IL8; STNFR; GRO; GRO-α; Amphiregulin; IL-1R4/ST2; TIMP1; TIMP2; UPAR; VEGF (Figure 12).
Therefore, this shows that two kinds of cell types all secrete a large amount of cytokines and the somatomedin that has critical function in developmental biology, tissue homeostasis, tissue repair and regeneration and blood vessel take place.This further confirms the multifunctionality of cell of the present invention in each therapeutic is used.
In addition, use mice teratoma to form and measure the safety of further checking cell of the present invention as indication.In these experiments, use six SCID mices.To be injected in the leg muscle of every SCID mice with the 25G sterile needle above the suspension of 200 ten thousand UCMC.Letting animals feed 6 months and assessment tumor form.Do not see that in these mices tumor forms (data not shown).This shows that cell of the present invention is safe and does not have the ability of optimum or other character tumor of any formation.
Also analyzed the ability of UCMC expressing human human leucocyte antigen (HLA) molecule.Divide the period of the day from 11 p.m. to 1 a.m when detecting major histocompatibility complex (MHC) I type, this analysis shows that there is (the testing result of arbitrary unit: 3201) in a large number in the HLA-A molecule, mean that cell is that HLA-A is male, and the not remarkable (testing result of arbitrary unit: 35), mean that cell is the HLA-B feminine gender of the expression of HLA-B molecule.These cells are also expressed HLA-G (referring to embodiment 19).Because HLA-B mainly works to the rejection of transplanting, this result represents that cell of the present invention not only is fit to autotransplantation, and suitable allograft.Cell detects positive to II type MHC molecule HLA-DR52, II type MHC molecule HLA-DRB4 is detected negative.Also find to exist HLA-DRB1 (0301/05/20/22).
Embodiment 4: cultivate ancestral cells in serum-free medium
Cultivate the UCMC cell at the DMEM that contains 10%FCS with in serum-free medium PTT-1, PTT-2 and PTT-3.Three kinds of culture medium PTT-1, PTT-2 and PTT-3 are prepared by one of inventor doctor Phan.In brief, these 3 kinds of culture medium do not contain hyclone or human serum, but contain different cytokines and somatomedin, as IGF, EGF, TGF-β, activator protein A, BMP, PDGF, transferrins and insulin.The somatomedin composition is different between culture medium, so that assess different growth characteristics.Cultivate following carrying out: the somatomedin and the cytokine of in basal medium, adding different proportion.UCMC thawed and in these culture medium, cultivated 10 days.Under optical microscope, monitor cell proliferation.The PTT-2 culture medium is melanocyte culture medium M154 and EpiLife (Ka Sikade biotech firm (Cascade Biologics Inc.), Oregon is USA) in the mixture of 3: 1 ratios.Culture medium 154 is aseptic, liquid tissue culture medium, with the preparation of 200 μ M calcium chloride, is used for the growth of ordinary people's epithelium keratinocyte.Culture medium 154 is to contain basal medium essential and non essential amino acid, vitamin, other organic compound, trace mineral and inorganic salt.It does not contain antibiotic, antifungal, hormone, somatomedin or protein.It is that HEPES and heavy carbonate are buffered, and has 5%CO 2Use in the atmospheric incubator of/95% air.
Figure 13 be presented at 4 kinds different cultivate basis set in UCMC well grow (Figure 13-1 is to Figure 13-5), wherein the form of UCMC cell is according to the difference of the cytokine that exists in each culture medium or somatomedin ratio or ratio and difference.On the contrary, the mesenchymal cell of bone marrow and adipose-derived undergrowth (Figure 13-6 and Figure 13-7) in these serum-free mediums.Therefore, the good growth of UCMC has shown the robustness of cell of the present invention and their high survival probability, shows that their growth characteristics are better than the mescenchymal stem cell in conventional source such as the mesenchymal cell of bone marrow derived and adipose-derived.With regard in this respect, it is worthy of note, in these experiments, used nothing (cattle) blood serum medium and most people's mesenchymal cell undergrowth in serum-free medium.Owing to reduced the risk of using hyclone to carry out cell culture and expansion, therefore in cell therapy, use cell of the present invention and described serum-free medium technology obviously favourable (though the cell growth has been carried out and optimized usually in the use of Ox blood serum for a long time, because of the propagation of zoonosis such as mad cow disease (bovine spongiform encephalopathy) increases the worry of using it).
Embodiment 5: the feature of umbilical cord epithelial stem cell and mescenchymal stem cell gene expression profile.
Use dna microarray to analyze the gene expression profile of umbilical cord (amniotic membrane) epithelial stem cell and mescenchymal stem cell.For this purpose, with UCMC and UCEC in growth medium in 37 ℃, 5%CO 2Following cultivation converges until 100%.Cell is replaced with fresh basal medium subsequently and was cultivated 48 hours again basal medium inter-syncization 48 hours.Gather in the crops total RNA and be sent to Silicon GeneticsMicroarray Service.Use GeneSpring 7.2 to carry out data analysis.Figure 14 has gathered full gene expression.UCEC expresses altogether 28055 genes and UCMC expresses 34407 genes altogether.There are 27308 eclipsed gene expressions in two kinds of cell types.747 gene pairs UCEC that express are unique, and 7099 gene pairs UCMC that express are unique.Selected genes of interest is listed in Figure 14.
Two kinds of stem cell types have been expressed 140 gene relevant with embryonic stem cell and fetal development: Nanog that further support cell of the present invention has embryonic stem cell sample characteristic; Alpha-fetoprotein; Pre B lymphocyte leukemia transcription factor 3; Laminin α 5; Carcinoembryonic antigen sample 1; Contain from hydrolytic enzyme territory 2; δ sample 3 (fruit bat (Drosophila)); Muscleblind sample (fruit bat); The GNAS complex locus; Carcinoembryonic antigen dependency cell adhesion molecule 3; Palmityl protein thioesterase 2; Pregnancy specific β-1-glycoprotein 2; Carcinoembryonic antigen sample 1; Embryonic ectoderm is grown; Parent embryo leucine zipper kinases; Chorion growth prolactin antagonist 2; Jaw box D3; Base edge albumen homologue (radical fringe homolog) (fruit bat); Kinesin family member 1B; Embryo's skeletal muscle myoglobulin heavy chain polypeptide 3; Cleft hand/foot deformity (adactylism toe) type 3; TEA territory family member 3; Laminin α 1; Chorion growth prolactin antagonist 1; Human placental lactogen; Corticotropin releasing hormone receptor 1; The thyrotropin embryo factor; Aromatic hydrocarbon receptor nuclear transposition albumen 2; Film frizzled related protein (Membrane frizzled-related protein); Neuregulin 1 ' XVI Collagen Type VI α 1; Neuregulin 1; Chorion growth prolactin antagonist 1 (human placental lactogen); The triple repetitive sequence rna binding proteins 1 of CUG; Chorion growth prolactin antagonist 1 (human placental lactogen) Bystin sample; MyoD family mortifier; Tretinoin induced protein 2; The GNAS complex locus; Pre B lymphocyte leukemia transcription factor 4; Laminin α 2 (congenital amyotrophy merosin); SMAD, the anti-DPP homologue 1 of parent (fruit bat); Has the mankind (H.sapiens) transcription sequence with protein pir:D28928 (mankind) D28928 pregnancy specific β-1 glycoprotein I B miscarriage property (fragment) medium similarity; Kinesin family member 1B; Rna binding protein Bruno sample 4 (fruit bat); The embryo and brain specific protein; The inductive growth inhibited albumen of gestation; SMAD, the anti-DPP homologue 5 of parent (fruit bat); Chorion growth prolactin antagonist 2; Adenyl cyclase reactivity polypeptide 1 (hypophysis); The cell adhesion molecule of carcinoembryonic antigen dependency; Layer adhesion egg α 3; Protein O-fucosyltransferase 1; Jagged 1 (alagille syndrome); Twist and form homologue 1 (fruit bat) gastrula; ELAV (fruit bat abnormal vision embryonic death) sample 3 (Hu antigens c); The thyrotropin embryo factor; 43 members 3 of solute carrier family; Inversion albumen (Inversin); Nephronophthisis 2 (baby); Embryo's inversion (inversion of embryonicturning) that overturns; Human inversion albumen (INVS) transcript variant 2mRNA; The human transcription sequence; Homeobox D8; Embryo Fyn dependency substrate; ELAV (fruit bat abnormal vision embryonic death)-sample 1 (Hu antigen R); Alkalescence contains helix-loop-helix territory B family 22; Ocytocin receptor; Teratoblastoma derivative growth factor 1; The Fms tyrosine kinase 1 (the saturating sex factor receptor of VEGF/blood vessel) of being correlated with; Adrenomedullin; The triple repetitive sequence rna binding proteins 1 of nuclear receptor co-activation factor 6-CUG; Twist and form homologue 1 (fruit bat) gastrula; Carcinoembryonic antigen relevant cell adhesion molecule 4; Receptor type Protein-tyrosine-phosphatase R; Acrg embryonic death (mice) Minimum Area is directly to congener; EPH receptor A3; δ sample 1 (fruit bat); The nose embryo LHRH factor; Transcription factor CP2 sample 1; Cleft hand/foot deformity (adactylism toe) type 3; Jagged 2; The human transcription sequence; Neuregulin 1; Cleft hand/foot deformity (adactylism toe) type 1; Solute carrier 43 family members 3; Hydroxyacylcoenzyme A dehydrogenase/3-ketoacyl coenzyme A thiolase/enoyl-CoA hydratase (three functional proteins) α subunit; Fucosyltransferase 10 (α (1,3) fucosyltransferase); Acrg embryonic death (mice) Minimum Area is directly to congener; Carcinoembryonic antigen relevant cell adhesion molecule 7; Nuclear phosphoprotein/nuclear phosphoprotein 2; IgG Fc fragment, receptor, transport protein, α; Twist and form homologue 1 (fruit bat) gastrula; The mankind are similar to sorting vacuole protein 35; Parent embryo 3 (LOC146485) mRNA; Contain from hydrolytic enzyme territory 2; Short-tail homologue T (mice); De-connect albumen and metalloproteinase domain 10; Ribosomal protein L 29; Endothelium peptide converting enzyme 2; ELAV (fruit bat abnormal vision embryonic death) sample 1 (Hu antigen R); Nourish albumen; Homeobox B6; Laminin α 4; Homeobox B6; Putative protein FLJ13456; Contain the NACHT that leucine enriches repetitive sequence and PYD 5; ELAV (fruit bat abnormal vision embryonic death) sample 1 (Hu antigen R); The differentiating embryonic cell transcription factor 1; PAPP-A, pappalysin 1; Secretion globin 1A family member 1 (uteroglobin); Parathyroid hormone sample hormone; Carcinoembryonic antigen relevant cell adhesion molecule 1 (biliary glycoprotein); Laminin α 1.
Two kinds of stem cell types have also been expressed thousands of and developmental biology, cell growth and differentiation, Cell Homeostasis, cell and tissue repair and the relevant gene of regenerating.This type of example of giving birth to factor and receptor thereof is as follows: (G-CSF, FGF, IGF, KGF, NGF, VEGF, PIGF, angiogenin, CTGF, PDGF, HGF, EGF, HDGF, TGF-β, activator protein and inhibin, Follistatin, BMP, SCF/c-Kit, LIF, WNT, SDF, oncostatin M, interleukin, chemotactic factor numerous in other); MMP, TIMP extracellular matrix (collagen, laminin, fibronectin, vitronectin, tenascin, integrate plain, cohere Dan Baijutang, the decorin gene polysaccharide, fibronectin, Dan Baijutang, the SPARC/ osteonectin, mucoprotein, lead albumen, glypican, cartilage is conjugated protein, extracellular matrix protein, hyaluronan, fine albumen, ADAMTS, disaccharidase catenin polysaccharide, coil basic mycoprotein, the desmosome composition, ICAM, cadherin, the connection albumen and numerous other); Cytokeratin.
Some group gene only occurs in UCMC.
Some group gene only occurs in UCEC.These genes are with following relevant: homeostasis (albumin; Calcium Sensing Receptor; Aquaporin 9; Breast fortune ferrum ferritin), (homeobox HB9 takes place in form; Epithelium V sample antigen 1), fetal development (relaxin 2; Carcinoembryonic antigen relevant cell adhesion molecule 8; Indole amine-pyrroles 2,3 dioxygenases; EPH receptor A3; The thyrotropin embryo factor; Pregnancy specific β-1-glycoprotein 1; α 3-laminin), extracellular space (surfactant alveolar dependency protein A 1; Pregnancy specific β-1-glycoprotein 1; Breast fortune ferrum ferritin; TGF-α; Albumin; FGF-23; S100 calbindin A9 (calgranulin B)), extracellular matrix (laminin β 4; Laminin α 3; Zona pellucida glycoprotein 4), structural molecule activity (chromosome 21 open reading-frames 29; Laminin α 3; Microtubule-associated protein 2; Laminin β 4; Keratin 6B; Ladinin 1; Keratin 6A; Closed albumen; Loricrin; Erythrocyte membrane protein band 4.1 (the chain elliptocytosis 1 of RH); Crystalline protein β A2; The crystalline lens structural protein; Contactin associated protein sample 4; Close protein 19; Putative protein LOC144501; Keratin 6E; Keratin 6L; Crystalline lens intergrate protein 2,19kDa), cytoskeleton (microtubule-associated protein 2; Erythrocyte membrane protein band 4.1 samples 5; Human trichohyalin (THH); Keratin 6B; Keratin 6A; The V sample antigen 1 of epithelium; Hook homologue 1 (fruit bat); Loricrin; Erythrocyte membrane protein band 4.1 (the chain elliptocytosis 1 of RH); Tropomodulin 1; MAP/ microtubule affinity is regulated kinases 1; Keratin 6E; Actin is in conjunction with LIM protein families member 2), cell adhesion molecule (2 type cadherins 19; Marrow/lymph or mixed stocker leukemia; Chromosome 21 open reading-frames 29; IRRE sample 2 compatriot (Kin); Laminin α 3; Sialoadhesin; CD84 antigen (human leucocyte antigen); Solubility galactoside associativity agglutinin (half agglutinin 2); Epithelium V sample antigen 1; CD96 antigen; The renal tubules tubulointerstitial nephritis antigen; Carcinoembryonic antigen relevant cell adhesion molecule 8; IL-18; Immunoglobulin superfamily member 1; Integrate plain β 8; Ornithine carbamoyl Ornithine arbamoyl transferring enzyme; Integrate plain β 6; Contactin associated protein sample 4; XVII Collagen Type VI α 1; Cadherin sample 26; Mucoprotein and cadherin sample), cell differentiation albumen (receptor type Protein-tyrosine-phosphatase Z polypeptide 1; α 3-laminin; CD84 antigen (human leucocyte antigen); EDRF2; The human erythrocyte is a differentiation correlation factor 2; Oncoprotein p73 sample; NB4 apoptosis/differentiation associated protein; Human PNAS-133; Similar to seven in absentia 2; Interleukin 24; Keratin 6B; Keratin 6A; Dehydrogenase/reductase enzyme (SDR family) member 9; Gap junction protein β 5 (connecting protein 31 .1); Iroquois homology frame protein 4; Homology frame 2 before the abdomen; Chemotactic factor (C-X-C motif) ligand 10; Tumor necrosis factor receptor super family member 17; Voltage-dependent calcium channel beta 2 subunit base; Parkinson disease (teenager autosomal recessive) 2 handkerchiefs gold albumen; (horny layer chymase) kallikrein 7; The homologue 2 of neurogliocyte disappearance; AP-2 α; Receptor type Protein-tyrosine-phosphatase Z polypeptide 1; TnT 1; Sciellin; Glucose amido (N acetyl group) transferring enzyme 2, the 1-branching enzyme; XVII Collagen Type VI α 1; Cytokine signaling repressor 2; Distal-less homeobox 1; Zygote stagnates 1; Interleukin II 0; Growth and differentiation factor 3; FGF-23; All-body configuration MMTV integration site family member 8A), extracellular: (chromosome 21 open reading-frames 29; Laminin α 3; Laminin β 4; Interleukin 24; Pregnancy specific β-1-glycoprotein 1; Chemotactic factor (C-X-C motif) ligand 11; Surfactant alveolar dependency protein A 1; Prepronociceptin; 5-hydroxy tryptamine (serotonin) receptor 3B; Carcinoembryonic antigen relevant cell adhesion molecule 8; Chemotactic factor (C-X-C motif) ligand 10; IL-18 (interferon gamma inducible factor); Breast fortune albumen; Albumin; Fas part (TNF superfamily member 6); Nicotine cholinoceptor β peptide 4; The Cathelicidin antibacterial peptide; Respiratory tract trypsin-like protease; S100 calbindin A9 (calgranulin B); TGF-α; KLK10; Kunitz type serine protease inhibitor 1; WNT1 inductivity signal pathway albumen 3; Relaxin 2; κ-interferon; Sozin β 103A; IL-20; Zona pellucida glycoprotein 4; Growth and differentiation factor 3; FGF-23; All-body configuration MMTV integration site family member 8A; Complement factor H relevant 5), grow protein (EPH receptor A3; NIMA (among the mitotic gene a from non-existent) associated kinase 2; Zinc finger protein 28 2; TANK-is in conjunction with kinases 1; The MRE11 meiosis 11 homologue A that recombinate; E2F transcription factor 2; Receptor type Protein-tyrosine-phosphatase Z polypeptide 1; The human mammary mRNA that expresses from X chromosome clones 161455; Laminin, α 3; V-myb myeloblastoma virus oncogene homologue (fowl) sample 1; G-protein signal instrumentality 11; Microtubule-associated protein 2; Transmembrane protein 16A; Adenoma polyp of colon 2; Homeobox HB9; Centromere protein F, 350/400ka (mitosin); CD84 antigen (human leucocyte antigen); EDRF2; Human erythrocyte's sample differentiation correlation factor 2; Oncoprotein p73 sample; NB4 apoptosis/differentiation associated protein; Human PNAS-133; Jaw box P2; Human stomach dependency differentially expressed protein YA61P (YA61); Tenascin N; Chromosome 6 open reading-frames 49; Zinc finger protein 46 2; Zinc finger protein 71 (Cos26); SRY (Sex determination zone Y) box 7; Start myeloid cell surface expression receptor sample 4; Interleukin 24; Pregnancy specific β-1-glycoprotein 1; Chondroitin sulfate proteoglycan 5 (neuroglycan C); Keratin 6B; Keratin 6A; Dehydrogenase/reductase enzyme (SDR family) member 9; Epithelium V sample antigen 1; β 5-gap junction protein (connecting protein 31 .1); G protein coupled receptor 51; Interferon regulatory factor 6; Neurotrophin 5 (neurotrophin 4/5); CD96 antigen; Iroquois homology frame protein 4; Interleukin 1 receptor sample 1; G-2 and S phase express 1; Nuclear receptor subunit family 2, E group membership 3; Homology frame 2 before the abdomen; Zinc finger protein 215; The sequence dna fragment of expressing on the chromosome 4 (uniqueness) 234; Carcinoembryonic antigen relevant cell adhesion molecule 8; Chemotactic factor (C-X-C motif) ligand 10; IL-18; Indole amine-pyrroles 2,3 dioxygenase enzymes; Albumin; Calcium Sensing Receptor (hypocalciuria hypercalcemia 1, serious introduction stage hyperparathyroidism); Fas part (TNF superfamily member 6); TNFR superfamily member 17; Calcium channel, voltage relies on, the beta 2 subunit base; Parkinson disease (autosomal recessive, teenager) 2, handkerchief gold albumen; (horny layer chymase) kallikrein 7; The homologue 2 of neurogliocyte disappearance; TGF-α; The thyrotropin embryo factor; AP-2 α (activation enhancer binding protein 2 α); KLK10; G protein signal instrumentality 7; Receptor type Protein-tyrosine-phosphatase Z polypeptide 1; Kunitz type serine protease inhibitor 1; WNT1 inductivity signal pathway albumen 3; Zic family member 3heterotaxy1 (the paired homologue of odd-, fruit bat); The TTK protein kinase; The slow TnT 1 of skeleton; Sciellin; The chain TGFB inducible factor 2 sample albumen of X; Kallikrein 8 (neuropsin/ovasin); Glucose amido (N acetyl group) transferring enzyme 2, the I-branching enzyme; Ankyrin repeat domain 30A; Relaxin 2; XVII Collagen Type VI α 1; The gene of differential expression in the prostate; Phosphatase and actin instrumentality 3; Cytokine signaling repressor 2; Nuclear receptor 4 subfamily A group memberships 3; Tonin (acyltransferase polypeptide-dipeptidase A) 1; Putative protein MGC17986; Distal-less homeobox 1; The long-lived homologue 3 (saccharomyces cerevisiae) of LAG1; Zygote stagnates 1; κ-interferon; IL-20; ICEBERG Caspase-1 mortifier; Growth and differentiation factor 3; FGF-23; Testis expressed sequence 15; All-body configuration MMTV integration site family member 8A; SRY (Sex determination zone Y) box 7; Carnitine lacks the indoor expression 1 of dependency; Prokineticin 1; Conjugated protein 3 samples 3 of CAMP response element; Caspase is called up domain family member 15; FLJ23311 protein).
Embodiment 6: umbilical cord liner epithelial stem cell (UCEC) directly is divided into the epiderm skin cutin and forms Cell
In order to be the epiderm skin keratinocyte, UCEC cell basis is used to cultivate the standard scheme cultivation of keratinocyte with umbilical cord epithelial stem cell UCEC cell differentiation.Cell separation technology as mentioned above.Subsequently with UCEC at serum-free keratinocyte growth medium KGM
Figure A20068004753500671
KGM
Figure A20068004753500672
-2 (Kang Baisi companies (Cambrex Corporation), New Jersey, USA), EpiLife (Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon, USA) or in Green ' s culture medium in the presence of the 3T3 mice embryonic feeder layer of handling through radiation or ametycin in 37 ℃, 5%CO 2Cultivate.Therefore, the differentiation of UCEC cellular morphology becomes similar human epidermic keratinocyte.Epithelial cell has similar morphology and can use and conventional easily changes fibroblast (with reference to figure 2) into the available culture medium of commerce under optical microscope.
Immunofluorescence analysis shows that the UCEC that cultivates also expresses epidermal keratinocytes molecular marker such as keratin, desmosome, hemi desmosome and basement membrane components (also referring to Figure 10, it shows that UCEC is qualified epithelial cell by expressing this multiple epithelioid cell sign substantially).Therefore these results show to be Skin Cell such as epidermal keratinocytes with umbilical cord epithelial progenitor cells/differentiation of stem cells of the present invention, and it can be used for wound healing and have the great potential that exploitation becomes the cultured skin equivalent.
Embodiment 7: use umbilical cord inner lining film tissue repeat organize outer planting to expand the umbilical cord epithelium to do carefully Born of the same parents and mescenchymal stem cell
Umbilical cord epithelial stem cell of the present invention and mescenchymal stem cell are expanded in the repetition outer planting of following use umbilical cord amnion tissue.In brief, at first day, will organize explant in 37 ℃, 5%CO 2Be seeded in (DMEM/10%FCS, EpiLife in the growth medium in tissue culture's ware , KGM
Figure A20068004753500674
KGM
Figure A20068004753500675
-2 or M171); Culture medium was changed once in per 2 days or 3 days.Cell grows beginning and moved continuously lasting 7 days from explant.After this, will organize explant to be transferred to another culture dish to allow the cell further growth.This process continues to carry out to have stoped further outer planting until the explant size decreases.In this article, should be noted that carrying out property of explant size diminishes until they too little outer plantings of can not further organizing that becomes because cell self-organizing explant grow and the process of moving out during, cell has produced the protease of digestion and degraded tissue.Figure 16 has schematically illustrated umbilical cord epithelial stem cell and the rapid and intensive expansion process of mescenchymal stem cell that obtains in this way that make.Therefore, from then on originally studies confirm that to originate obtains high yield UCMC and UCEC cell, and this has further reflected high survival probability and the growth promotion feature of comparing these cells with the stem cell of the cell in other source such as bone marrow or adipose-derived.In addition, as solid tissue, the outer planting technology of successful repetition used herein confirms and can be not only and extract cell of the present invention certain part equably from complete tissue.This can obtain the cell of maximum, and it can be through passing at the low for deriving and not making cell through causing repeatedly going down to posterity of cell degradation.
Embodiment 8: liner mesenchymal cell (UCMC) directly is divided into dermis of skin and becomes fiber between umbilical cord Cell
In order to be the dermis of skin fibroblast, it is cultivated according to being used for fibroblastic standard scheme umbilical cord mesenchymal stem cells UCMC cell differentiation.Described in cell separation technology such as the top embodiment 6.Subsequently UCMC is cultivated in DMEM or commercial available fibroblastic growth culture medium (FGM).Therefore, the differentiation of UCMC cellular morphology becomes similar human dermis fibroblast.Mesenchymal cell has similar form and can use and conventional is transformed into fibroblast (with reference to figure 3) easily with the available culture medium of commerce under optical microscope.
Embodiment 9:UCEC directly is divided into the epiderm skin keratinocyte
With embodiment 6 similar methods in, press among the embodiment 2 the separation umbilical cord amniotic membrane epidermis ancestral cells of describing (UCEC).For UCEC is divided into epidermal keratinocytes, at keratinocyte culture medium (EpiLife
Figure A20068004753500681
Or KGM
Figure A20068004753500682
) in cultured cell be that DMEM/10%FCS continues to form in 3 days cuticular cellulose up to 100% converging (Figure 17-A show cultivating 5 days after), changing culture medium then.(wherein having described the photo of two experiments that are called as " UCEC-10 " and " UCEC-17 ") as Figure 17-A demonstration, after in DMEM/10%FCS, cultivating, UCEC has been divided into epidermal keratinocytes, and it forms cellular layer (photograph taking of Figure 17-A is after 10 days).Therefore these results provide further evidence for the totipotency of cell of the present invention.
Embodiment 10:UCMC directly is divided into osteoblast
Press the separation umbilical cord amniotic mesenchymal cell of describing among the embodiment 2 (UCMC).For UCMC is divided into osteoblast, cultured cell converges up to 100% in DMEM/10%FCS, cultivates 48 hours in the hungry culture medium of the DMEM of serum-free then again.It is preceding to carry out Feng Kesa dyeing (osteocyte dyeing) at pair cell, and UCMC is placed 4 weeks of osteogenesis inducing culture.The osteogenesis inducing culture contains DMEM/10%FCS; 1% antibiotic (streptomycin and penicillin)/antifungal (amphotericin); 0.01 μ M 1,25-dihydroxyvitamin D3,50 μ M ascorbic acid-2-phosphate, 10mM beta-glycerophosphate, 1% antibiotic (streptomycin and penicillin)/antifungal (amphotericin).
As showing among Figure 17 B, the formation that the Feng Kesa of the UCMC cell of cultivation in the osteogenesis inducing culture dyes joint among the colour specification UCMC, therefore UCMC is divided into osteoblast, and in the UCMC that is untreated, do not show this type of differentiation, the described UCMC of being untreated is as negative control, cultivates in other condition identical but do not have among the inductive DMEM/10%FCS.As other negative control, from the dermal fibroblast of 8 months big donors and from the fibroblasts in keloid of 20 years old donor with inductive or not inductive UCMC the same terms under cultivate.Two kinds of cell types use Feng Kesa dyeing all not produce positive findings, and this is the totipotency of UCMC of the present invention, and therefore also is divided into for example osteoblastic further evidence.
Embodiment 11:UCMC directly is divided into adipose cell
Press the separation umbilical cord amnion mesenchymal ancestral cells of describing among the embodiment 2 (UCMC).For UCMC is divided into adipose cell, cultured cell converges up to 100% in DMEM/10%FCS, cultivates 48 hours in the hungry culture medium of the DMEM of serum-free then again.Before carrying out oil red O stain, UCMC is placed 4 weeks of lipogenesis inducing culture.The lipogenesis inducing culture contains DMEM/10%FCS; 1% antibiotic (streptomycin and penicillin)/antifungal (amphotericin); 0.5mM isobutyl group-methylxanthine (IBMX), 1 μ M dexamethasone, 10 μ M insulins and 200 μ M indomethacins.
As showing among Figure 17 C, the oil red O stain of the UCMC cell of cultivation in the lipogenesis inducing culture is represented the fat accumulation among the UCMC, therefore UCMC is divided into adipose cell, and in the UCMC that is untreated, do not show this type of differentiation, the described UCMC of being untreated is as negative control, cultivates in other condition identical but do not have among the inductive DMEM/10%FCS.As other negative control, from the dermal fibroblast of 8 months big donors and from the fibroblasts in keloid of 20 years old donor with inductive or not inductive UCMC the same terms under cultivate.Two kinds of cell types all do not produce positive findings in oil red O stain, this is the totipotency of UCMC of the present invention, and are divided into for example further evidence of adipose cell yet.
Embodiment 12: use UCMC and UCEC to produce skin with collagen as extracellular matrix component The method of skin equivalent
In order to produce exemplary skin equivalent of the present invention, use 6 hole tissue cultures dish, it contains the support (Transwell that is made by 3 μ m porous polycarbonate films
Figure A20068004753500701
, Corning Incorporated (CorningIncorporated), Massachusetts, USA).This type of dish can be from the AG of Vitalis company (Vitaris AG), Baar, and Switzerland obtains.For collagen ECM layer, use the collagen (1.0-1.7mg/ml) of the sterilized rat afterbody acid-extraction in 0.05% acetic acid.Other component that is used for acellular ECM culture medium and cell ECM culture medium is 10 * minimum essential medium, it has EarleShi salt (Ji Buke BRL company (Gibco-BRL), Maryland, USA), L-glutaminate (200mM, Ji Buke BRL company (Gibco-BRL), Maryland, USA), sodium bicarbonate (71.2mg/ml), DMEM (Ji Buke BRL company (Gibco-BRL), Maryland, USA), hyclone (FBS, Ji Buke BRL company (Gibco-BRL), Maryland, USA).Cell culture medium PTT-4 and PTT-7 for different, use following component:
PTT-4:90% (v/v) CMRL 1066 and 10% (v/v) tire calf serum.
PTT-7:99.4% (v/v) EpiLife
Figure A20068004753500702
, 0.2% (v/v) insulin, 0.2% (v/v) transferrins, 0.2% (v/v) Monohydrated selenium dioxide and 10ng/ml epidermal growth factor (EGF).
Press among the embodiment 2 and 7 separating of describing and cultivate umbilical cord amnion mesenchymal stem cell and epithelial stem cell (UCMC and UCEC).Figure 18 b shown cultivated UCMC (cultivating after 7 days) in PTT-4, and Figure 18 a has shown cultivate UCEC (cultivating after 7 days) in PTT-7.
All said components all should remain on ice.In order to produce the extracellular matrix (acellular ECM) that does not contain cell, acellular ECM nutrient media components is mixed by the order of enumerating in the table 1.The color of culture medium solution should be faint yellow to light red, and any excessive variation on the color can represent to make collagen not become the pH of gel.After acellular ECM culture medium mixes, add this culture medium of 1ml to each support, guarantee the whole bottom of mixture coating stent.In case toppled over collagen gel, just should not be in the incubator (37 ℃, 5%CO 2) disturb.
In case acellular ECM polymerization (after collagen gel was poured support into, it needed about 0.5 to about 12 hours usually), the UCMC in just can trypsinization cultivating, and it thoroughly is resuspended in the PTT-4 cell culture medium, reaching final concentration is 5 * 10 5Cell/ml prepares to mix with collagen solution.
For cell ECM culture medium (referring to table 1), all components all should remain on ice.In order to produce the extracellular matrix (cell ECM) that contains cell, cell ECM nutrient media components is mixed by the order of enumerating in the table 1.Should in the end add the UCMC that is dissolved among the PTT-4, thereby guarantee mixing, thereby cell can not destroyed by alkaline pH by interpolation collagen neutralization.Mixing also adds 3ml in support, and allow its in incubator (37 ℃, 5%CO 2) the one-tenth gel.When gel is pink and solidifies (after 2 to 24 hours), they are covered and hatched 4 to 7 days with 3 to 4ml PTT-4, shrink stable and complete up to gel.It is fibroblast (Figure 18 b) that the PTT-4 culture medium allows cell differentiation.Another is fit to differentiation UCMC is that fibroblastic culture medium is described in embodiment 8.During interval, the volume of substrate reduces 50 times.Should change culture medium in per 2 days.
The ready UCEC of trypsinization, thus can be with high among the 50 μ l PTT-7 to 1 * 10 6Individual cell bed board is in each support.Cell suspension can be placed into the flat-top sample part of central protrusion of the collagen gel of contraction with the automatic pipettor of 200 μ l.
When UCEC attaches on the fibroblast layer that has formed, do not touch flat board in 2 to 3 hours in incubator.After this incubation period, PTT-7 is joined on the top of UCEC (2ml) in the hole (4ml), cultivation reaches 7 days.Should change culture medium in per 2 days.It is that (Figure 18 a) for keratinocyte that the hatching of UCEC and PTT-7 allows cell differentiation.The culture medium that another suitable differentiation UCEC is a keratinocyte is the culture medium of describing in embodiment 6.
In this, in the hole, skin equivalent (CSE-1) is grown in gas-liquid interface, uses the high calcium (0.6mM) that is dissolved in the PTT-7 culture medium (only 1.5-2ml), and cultivation reaches 10 days.Should change culture medium in per 2 days.
Skin equivalent (CSE-1) is ready for histology and electron-microscopic analysis now.The growth that should be noted that CSE-1 changes with the different of UCMC strain with the UCEC that uses in the method.Can see by the photo from Figure 19, form skin corium of forming by fibroblast and the epidermal area of forming by keratinocyte.Photo among Figure 20 a has shown after being promoted to liquid-vapor interface, the appearance of CSE-1.Figure 20 b has shown the outward appearance of accumulative UCMC in the collagen lattice of CSE-1.
The component of table 1:ECM culture medium
6ml is acellular, and ECM cultivates 18ml cell ECM culture medium (3ml/
Base (1ml/ support) Support)
10×DMEM 0.59ml 1.65ml
L-glutaminate 0.05ml 0.15ml
Hyclone 0.6ml 1.85ml
Sodium bicarbonate 0.17ml 0.52ml
Collagen 4.6ml 14ml
UCMC - In the PTT-4 of 1.5ml culture medium 5 * 10 5Individual cell
Embodiment 13: use UCMC and UCEC to produce the method for skin equivalent
Generally speaking, the following example is undertaken by the mode identical with embodiment 12.Yet, opposite with embodiment 12, do not use collagen as ECM (described collagen in embodiment 12, before inoculation UCMC is in the support, being incorporated in the support).In the present embodiment, UCMC cell self secretion type i collagen forms three-dimensional cellular layer, thus the extra collagen layer of unnecessary use.Realize by in cell culture medium PTT-4, adding ascorbic acid by UCMC cells produce collagen.
At first, press the results UCMC cell of describing among the embodiment 12, UCEC is by 1 * 10 5The concentration of individual cell/ml is seeded on the support, and hatch 3 days (37 ℃, 5%CO 2).
After 3 days, in culture medium PTT-4, replenish 50 μ g/ml ascorbic acid.And then 2 weeks of incubated cell (37 ℃, 5%CO 2).Under these conditions, UCMC deposits type i collagen with the oneself, and forms three-dimensional cellular layer, the extracellular matrix that produces separately among its simulation embodiment 12.
In order to produce epithelial layer, carry out the identical experiment of having described among the embodiment 12.
Skin equivalent (CSE-2) is ready for histology and electron-microscopic analysis now.The growth that should be noted that CSE-2 changes with the different of UCMC strain with the UCEC that uses in the method.The appearance of CSE-2 can have been shown from the photo Figure 21 a after being promoted to liquid-vapor interface.Figure 21 b has shown the form of accumulative UCMC in the collagen lattice of CSE-2.
Embodiment 14: use UCMC, HUVEC and UCEC with the method for producing the skin equivalent as the collagen of extracellular matrix component
Generally speaking, the following example uses and embodiment 12 described identical component and programs.
Press among the embodiment 2 and 7 separating of describing and cultivate mesenchyme and epithelial stem cell (UCMC and UCEC) from the umbilical cord amniotic membrane.Use collagenase to cultivate people's umbilical blood vessels endotheliocyte (HUVEC) from cord vessels.In brief, remove whole blood with PBS flushing cord vessels.Clamping umbilical cord end: 0.5% collagenase solution is expelled in the cord vessels, and incubated at room 20 minutes.Then, remove clip, and (Kang Baisi company (Cambrex Corporation), New Jersey USA) wash cord vessels, thereby collect the HUVE-cell suspending liquid with the EGM culture medium that contains 10% tire calf serum (FCS).The centrifuge cell suspension, the collecting cell precipitation is then EGM culture medium (Kang Baisi company (Cambrex Corporation), New Jersey, USA) the middle cultivation.
Experimentizing not long ago, and in incubator (37 ℃, 5%CO 2) (Kang Baisi company (Cambrex Corporation), New Jersey cultivate the HUVEC cell USA) or in the M131 culture medium in the EGM culture medium.The immunohistochemical analysis of the usage factor VIII related antigen or the von Willebrand factor is verified the cell purity (data not shown) of these cells.In brief, HUVEC is seeded in reaches 80% up to them on the coverslip and converge.Then, they are fixed and, resist with two of peroxidase conjugated then and hatch with the antibody incubation of the anti-or anti-von Willebrand factor of anti-Factor IX.The percentage ratio of the cell of the Factor IX that the number of positive cell has been expressed as labelling or the von Willebrand factor.Examine under a microscope positive cells to check their purity.
The all components that experiment is used all should remain on ice.In order to produce the extracellular matrix (acellular ECM) that does not contain cell, acellular ECM nutrient media components is mixed by the order of enumerating in the table 2.Other details is referring to the description in the experiment 12.
In case acellular ECM polymerization is (after collagen gel is poured support into, it needs about 0.5 to about 12 hours usually), UCMC and HUVEC in just can trypsinization cultivating, and it thoroughly is resuspended in PTT-4 (being used for UCMC) and M131 (being used for HUVEC) cell culture medium, reaching final concentration is 5 * 10 5Cell/ml (UCMC/HUVEC-1: 1 mixture), prepare to mix with collagen solution.
For cell ECM culture medium (referring to table 2), all components all should remain on ice.In order to produce the extracellular matrix (cell ECM) that contains cell, cell ECM nutrient media components is mixed by the order of enumerating in the table 2.Should in the end add the UCMC/HUVEC mixture, thereby guarantee mixture, thereby cell can not destroyed by alkaline pH by interpolation collagen neutralization.Mixing also adds 3ml in support, and allow its in incubator (37 ℃, 5%CO 2) the one-tenth gel.When gel solidifies (after 30 minutes), they were covered and hatch 4 to 7 days with 3 to 4ml PTT-4/M131 (1: 1), shrink stable and fully up to gel.It is fibroblast that the PTT-4 culture medium allows cell differentiation.During interval, the volume of substrate reduces 50 times.Should change culture medium in per 2 days.
Trypsinization UCEC when being ready to, thus can be with high among the 50 μ l PTT-7 to 1 * 10 6Individual cell is taped against in each support.Cell suspending liquid can be placed into the flat-top sample part of central protrusion of the collagen gel of contraction with 200 μ l automatic pipettors.
When UCEC attaches on the fibroblast layer that has formed, do not touch flat board in 2 to 3 hours in incubator.After this incubation period, add PTT-7 at the top of UCEC (2ml) and arrive in the hole (4ml), cultivation reaches 7 days.Should change culture medium in per 2 days.It is keratinocyte that the hatching of UCEC and PTT-7 allows cell differentiation.
In this, in the hole, skin equivalent (CSE-3) is grown in gas-liquid interface, uses the high calcium (0.6mM) that is dissolved in the PTT-7 culture medium (only 1.5-2ml), and cultivation reaches 10 days.Should change culture medium in per 2 days.
Skin equivalent (CSE-3) is ready for histology, copolymerization Jiao, ultramicroscope and immune analysis now.The growth that should be noted that CSE-3 changes with the different of UCEC strain with the UCMC that uses in the method, HUVEC.
The component of table 2:ECM culture medium
ECM culture medium that 6ml is acellular (1ml/ support) 18ml cell ECM culture medium (3ml/ support)
10×DMEM 0.59ml 1.65ml
L-glutaminate 0.05ml 0.15ml
Hyclone 0.6ml 1.85ml
Sodium bicarbonate 0.17ml 0.52ml
Collagen 4.6ml 14ml
UCMC - In the PTT-4/M131 of 1.5ml mixture culture medium 5 * 10 5Individual cell
Embodiment 15:UCEC and UCMC are divided into β-islet-like cells respectively
Press mesenchyme and the epithelial stem cell (UCMC and UCEC) of the separation of description among the embodiment 2 from the umbilical cord amniotic membrane.
In PTT-4 culture medium (composition of the PTT-4 that sees above (90% (v/v) CMRL1066,10% (v/v) FCS)) or PTT-10 culture medium, hatch UCMC.The PTT-10 culture medium contains 99.4% (v/v) CMRL-1066 and 0.2% (v/v) insulin, 0.2% (v/v) transferrins and 0.2% (v/v) Monohydrated selenium dioxide.
In PTT-7 culture medium (composition of the PTT-7 that sees above) or PTT-5 and PTT-6 culture medium, hatch UCEC.The PTT-5 culture medium contains 98.8% (v/v) CMRL-1066,0.4% (v/v) insulin, 0.4% (v/v) transferrins, 0.4% (v/v) Monohydrated selenium dioxide and 10ng/ml epidermal growth factor (EGF).The PTT-6 culture medium contains 99.4% (v/v) M171,0.2% (v/v) insulin, 0.2% (v/v) transferrins, 0.2% (v/v) Monohydrated selenium dioxide and 10ng/ml epidermal growth factor (EGF).
By adding the 1mM nicotiamide in the cell culture medium separately of cultivating cell, the differentiation of inducing UCEC and UCMC respectively.Should change cell culture medium in per 2 or 3 days.
Examine under a microscope outward appearance (Figure 23 of β-islet-like cells; There is being or do not having inducer, i.e. UCEC among the PTT-10 of nicotiamide).Collect β-islet-like cells and be used for microscopic analysis.In order to determine whether the cell that the method according to this invention obtains is β-islet-like cells really, use is at Technical Manual of StemCell Technologies Inc., title: In-vitrodifferentiation of murine ES cells into pancreatic islet-like clusters, or " Epithelial-to-mesenchymal transition generates proliferative human isletprecursor cells ", Science, 2004, a kind of method of describing in 306, the 2261-2264 pages or leaves.
Insulin-inductive the alternative method of production cell differentiation of inducing UCEC (CLEC) has hereinafter been described.Hatch UCEC with one of above-mentioned culture medium (PTT-5, PTT-6 or PTT-7 culture medium).In order to induce UCEC to be divided into insulin-production cell, with these cellular exposure the ESCult culture medium (available from Stemcell Technologies Inc. (CA) (StemCell Technologies Inc.), Vancouver, Canada) or in the BBRC06 culture medium 7 days.
(Stemcell Technologies Inc. (CA) (StemCell Technologies Inc.), Vancouver Canada) contains 100 milligrams/ml nicotiamide to the ESCult culture medium, adds with 20ml/l B27 and 10ml/l N2 additive.
The BBRC06 culture medium contains serum-free DMEM/F12 culture medium, it has the 17.5mM glucose, and there is a nicotiamide 10mM, activator protein-A 2nM, Exendin-410nM, hepatocyte growth factor 100pM and pentagastrin 10nM (Sigma-Alder Riker Inc. (Sigma-Aldrich), Missouri, USA) and B-27 serum-free additive, N-2 additive (Stemcell Technologies Inc. (CA) (StemCell Technologies Inc.), Vancouver, Canada), with 1% penicillin/streptomycin 5000U/L (Biochem.Biophys.Res.Commun.2006, March24; 341 (4) 1135-40 pages or leaves).
After in ES Cult culture medium or BBRC06 culture medium, hatching 7 days, use from QIAGEN
Figure A20068004753500761
(Hilden, Germany) is used for the Rneasy of RNA extraction and purification
Figure A20068004753500762
Extract test kit and gather in the crops total RNA, single sample is measured through RT-PCR and is detected the insulin gene expression.Use the insulin primer as positive control, it is described in Timper K., people such as Seboek, and Biochem.Biophys.Res.Commun.2006, March 24; In 341 (4) the 1135-40 pages or leaves.
Figure 25 has shown that this substitutes the result of abductive approach.Figure 25 is presented under the inducing of ES Cult culture medium or BBRC06 culture medium, the insulin expression in a plurality of UCEC samples.Be appointed as CLEC25,28 and represent different samples from different umbilical cord donors with 30 sample.Be appointed as the sample of CLEC30 (1) and CLEC30 (2) and represent the duplicate of same sample.Therefore, having proved that UCEC has is divided into the potentiality that can be used for treating glycosuria insulin-production cell not.
That embodiment 16:UCEC is divided into is mucoprotein-produce cell
Press the epithelial stem cell (UCEC) of the separation of description among the embodiment 2 from the umbilical cord amniotic membrane.
Isolating UCEC be grown in PTT-5 or the PTT-6 cell culture medium (37 ℃, 5%CO 2), its composition has been described among the embodiment 15.The surprising discovery of the present invention uses PTT-5 or PTT-6 cultivation UCEC to make UCEC production mucoprotein (referring to Figure 22 a).Move liquid and changing in the process of culture medium the viscosity of observation of cell culture supernatants.
Mucoprotein grumeleuse test: mucoprotein grumeleuse test is to the mucoprotein quality of the UCEC production of cultivating among PTT-5 or the PTT-6 and the assessment of quantity.This test is gone back general description at CorfieldA.P., Glycoprotein method and protocols:The Mucins, 29-65 page or leaf .Humana Press 2000; Gatter R.A. and Schumacher R.H., A practicalhandbook of join fluid analysis, 59-63 page or leaf, Lea﹠amp; Febiger is in 1991.In this test, in UCEC cell culture condition culture medium, spray into the 7N glacial acetic acid.Acetic acid causes mucoprotein formation grumeleuse.Contain common mucoprotein UCEC cell culture condition culture medium show as have closely, the clear liquid of thickness grumeleuse.
For the mucoprotein amount that quantitative UCEC produces, carry out SDS-PAGE and coomassie subsequently dyeing (referring to Figure 22 b).For SDS-PAGE, use 100kDa mwco membrane Centrisart
Figure A20068004753500771
I (Sai Duolisi company (Sartorius AG), Germany) concentrates the 2.5ml cell culture medium of collecting from the UCEC cell culture.Sample on the spissated supernatant of 100 μ l is used for electrophoresis in 6%SDS-PAGE.Then with the coomassie gel that dyes, and take pictures.
Embodiment 17:UCMC directly is divided into chondrocyte (cartilage forms system)
Growing to UCMC is osteoblast similar (referring to embodiment 10), and they also have the ability of growing for the chondrocyte of forming cartilage.
To be cartilage in order growing, to press the separation umbilical cord amniotic mesenchymal cell of describing among the embodiment 2 (UCMC).For UCMC is divided into osteoblast, cultured cell converges up to 100% in DMEM/10%FCS.Then they are exposed to and have replenished 10ng/ml transforming growth factor-beta 3 (TGF-β 3; R﹠amp; (the R﹠amp of d system company; D Systems), Minneapolis, USA), 100nM dexamethasone, 50mg/mL ascorbic acid, 100mg/mL Sodium Pyruvate, 40mg/mL proline (Sigma-Alder Riker Inc. (Sigam-Aldrich), Missouri, USA) in the PTT-5 culture medium, continued for 4 weeks.Cellular layer is with alcian blue dyeing (its dyeing acid glycosamine glycan and mucopolysaccharide).
Compare (Figure 24 A) with the contrast that does not wherein show this type of differentiation, chondrocyte or the cartilage observed in Figure 24 B with the alcian blue positive staining form system.Contrast is made up of untreated UCMC, and it cultivates in other condition identical but among the inductive DMEM/1%FCS of additional PTT-5 of no use (above).The result shows that UCMC has the potentiality of the chondrocyte that forms cartilage, is used for repair of cartilage and regeneration.By using for example TissueFleece of support (Austria hundred special companies (Baxter AG), Austria; Be described among the embodiment 21) or hydrogel cell can be used for body.
Embodiment 18:UCMC directly is divided into dopamine-production cell
This embodiment has described UCMC and has been divided into dopamine and tyrosine hydroxylase (TH)-production cell.
For the growth of dopamine and TH, press the separation umbilical cord amniotic mesenchymal cell of describing among the embodiment 2 (UCMC).
In 100mm tissue culture ware, cultivate UCMC, and in the PTT-4 culture medium, keep and converged up to 80% in 5 days with the density of 200000 cells.Then with cellular exposure in the PTT-2 culture medium 48 hours.After hatching, the collecting cell culture supernatants is used for analyzing.The PTT-2 culture medium is M154 (melanocyte culture medium) and EpiLife
Figure A20068004753500782
(Ka Sikade biotech firm (Cascade BiologicsInc.), Oregon is USA) in the mixture of 3: 1 ratios.
(supernatant carries out Western trace or immunohistochemistry (IHC) analyses as mentioned) to UCMC cell or conditioned medium.Figure 26 A shows that CLMC (UCMC) emiocytosis TH is in conditioned medium.When being exposed to the PTT-2 culture medium, observe more TH secretion in the 4th and 5 roads.Figure 26 B shows the dopamine secretion of CLMC (UCMC) cell.Figure 26 C shows negative control.Coax (coax) these UCMC cells with dopamine.They keep not differentiation, but can detect a large amount of dopamine.Expression: UCMC has the potentiality of producing dopamine and dopamine precursor-TH.
Embodiment 19:UCMC and UCEC are divided into HLA-G-and produce cell
This embodiment has described UCMC and UCEC is divided into HLA-G-production cell.HLA-G is a HLA I class antigen, mainly expresses in Placenta Hominis, and supposition may protect fetus not by its mother's immune system attack.HLA-G is special HLA, relates to the disease and the disease of panimmunity mediation, as organ-, cell transplantation and autoimmune disease.The example of this type of autoimmune disease is multiple sclerosis, rheumatoid arthritis, type i diabetes, psoriasis, thyroid disease, systemic lupus erythematosus (sle), scleroderma or celiac disease.Reported the specific variants relevant with the risk of miscarriage and preeclampsia.The HLA typing is crucial for the donor and the receptor coupling of bone marrow transplantation; The donor that use matches has increased survival and has reduced graft antagonism host's disease.The HLA typing is for solid organ transplantation, and the application in other field also is important.
For the growth of HLA-G, by separation umbilical cord amniotic epithelial cells and the mesenchymal cell (being respectively UCEC and UCMC) described among the embodiment 2.
From UCMC and total RNA of UCEC cell harvesting and conditioned medium, it is carried out RT-PCR and Western engram analysis.Figure 27 A is presented at the expression of the HLA-GmRNA among UCMC and the UCEC.The positive control that uses the JAR trophoblastic cell to express as HLA-G.Figure 27 B shows that UCEC secretion HLA-G protein is in conditioned medium.
This experiment show for the first time inmature UCEC (
Figure A20068004753500791
UCEC) express and produce HLA-G.For the production of HLA-G, the specificity of UCEC is induced not necessarily.HLA-G-produces UCEC and the UCMC cell has good immunosuppressant character.These cells are reduced immunogenicities, are the good candidate of allograft.
Embodiment 20: use UCMC to induce old and feeble skin keratin to form the propagation of cell (asK)
In the following example, showed how UCMC can induce old and feeble skin keratin to form the propagation of cell (asK) and human dermis fibroblast (NF).
Press the mesenchymal cell (UCMC) of the separation umbilical cord amniotic membrane of describing among the embodiment 2.Cultivating UCMC in the PTT-4 culture medium converges up to 80%.Then, harvesting and centrifugal about 10 minutes at 1200rpm.After centrifugal, the ratio in the low infiltration of per 800 ten thousand cell 1ml in cell precipitation adds low infiltration.Total cell lysate was centrifugal 30 minutes of 4 ℃ of 4000rpm.Collect the clarification phase of " UCMC extract " and be stored in-30 ℃.From the skin (patient more than 60 years old) of chronological age, separate old and feeble skin keratin and form cell (asK).Density with 4000 cells/well in 24 hole flat boards is planted the ask cell, and maintains growth EpiLife
Figure A20068004753500792
(Ka Sikade biotech firm (CascadeBiologies Inc.), Oregon, USA) 24 hours in the culture medium.Then with the ask cellular exposure at basic EpiLife
Figure A20068004753500793
(Ka Sikade biotech firm (Cascade Biologies Inc.), Oregon USA) continue 48 hours, press different dilution factors then at basic EpiLife in the culture medium Add the UCMC extract of 2.5%, 5%, 10% and 20% dilution in the culture medium.Measure at different interval implementation criteria MTT.Figure 28 A is presented at the 6th and 7 day with the inductive ask cell proliferation of the UCMC extract of 2.5%, 5% and 10% concentration." contrast " sample column representative of showing among Figure 28 A maintains the basic EpiLife that does not add UCMC extract or somatomedin
Figure A20068004753500795
The skin keratin of the aging in the culture medium forms cell.The skin keratin that term " asK10 (1) p3 " refers to pass the aging in 3 generations forms cell." positive " sample column representative of showing among Figure 28 A maintains growth medium (the basic EpiLife of the optimal conditions that does not have the UCMC extract
Figure A20068004753500801
Culture medium+somatomedin) cell in.Similar, the fibroblastic different cell line of human dermis (being respectively NF125 and NF119) is handled as above-mentioned usefulness " UCMC extract ".These result of experiment are presented among Figure 28 B and Figure 28 C.
Because " UCMC extract " skin keratin is formed cell and fibroblast has positive influence, can prepare extract and be used to promote wound healing, skin repair, regeneration and rejuvenation (rejenuvation).
Embodiment 21: use different timbering materials to be used for the cultivation of UCMC
In first experiment, by the UCMC cell preparation mescenchymal tissue equivalent (MTE) of in collagen lattice, breeding.Cultivate construct altogether in order to prepare organotypic, MTE is transferred to (Corning Incorporated (Corning Inc.)) in the Transwell insert.CLEC or application on human skin keratinocyte are pressed 100,000 cell/cm 2Density plant on the MTE, and maintain in the EpiLife culture medium.Then organotypic is cultivated construct altogether and rise to liquid-vapor interface, continued for 3 weeks.Then construct is carried out H﹠amp; E dyeing is used for histologic analysis (referring to Figure 29 A and 29B).
Carry out second experimental results show that UCMC inoculates into the biomaterial scaffolds that is purchased TissuFleece for example
Figure A20068004753500802
(Austria hundred special companies (Baxter AG), Austria), INTEGRA BilayerMatrix Wound Dressing TM(knob sieve scientific company (Neuro Sciences), New Jersey, USA), BoneSaxe
Figure A20068004753500803
(U.S. Stryker Corp. (Stryker Inc.), MI, USA) or the ability among the Synthese (AO).
TissuFleece
Figure A20068004753500804
E (Austria hundred special companies (Baxter AG), the support that active component Austria) is made up of the exsiccant Corium Equi skin of freedom collagen.1cm 2TissuFleece
Figure A20068004753500805
The natural collagen fibril that contains 2.8mg.General, collagen causes platelet aggregation with contacting of blood, then in a large number attached on the collagen stroma, and cracked and release coagulation factors, described coagulation factors causes fibrinous formation with blood plasma factor.Collagen stroma increases grumeleuse in addition.Since its structure, TissuFleece
Figure A20068004753500806
The collagen net can absorb big quantity of fluid.Only, absorb UCMC by this mechanical process.Therefore, quicken the formation of granulation tissue.
INTEGRA Bilayer Matrix Wound Dressing TM(hereinafter only be called Integra
Figure A20068004753500807
) be to comprise the porous matrix of crosslinked beef tendon collagen and glycosaminoglycans and the support of semipermeability polysiloxanes (siloxanes) layer.The forfeiture of semipermeability silicone film control steam provides the flexible of wound coverage surface to adhere to, for device has added enhanced tearability.Collagen-glycosaminoglycans biodegradable matrices provides and has been used for the support that cell is invaded and blood capillary is grown.
BoneSaxe
Figure A20068004753500811
(U.S. Stryker Corp. (Stryker Inc.), MI, USA) support of forming by calcium phosphate ceramic.
ChronOS
Figure A20068004753500812
(Lin Saisi company (Synthes GmbH), the Switzerland) support of forming by bata-tricalcium phosphate.
In order to test, press the mesenchymal cell (UCMC) of the separation umbilical cord amniotic membrane of describing among the embodiment 2.Cultivating UCMC in the PTT-4 culture medium converges up to 80%.Harvesting and under 1200rpm centrifugal about 5 minutes to obtain cell precipitation.For at TissuFleece
Figure A20068004753500813
, Integra
Figure A20068004753500814
BoneSave
Figure A20068004753500815
(U.S. Stryker Corp. (Stryker Inc.), MI, USA) and ChronOS
Figure A20068004753500816
(Lin Saisi company (Synthes GmbH), Switzerland) UCMC is gone in middle inoculation, and the ratio of cell precipitation in 800 ten thousand cells of every 1ml culture medium is resuspended in the PTT-4 culture medium.Then with 100000 cell/cm 2Density the UCMC cell suspending liquid is planted in TissuFleece
Figure A20068004753500817
Or Integra
Figure A20068004753500818
On the layer.BoneSave
Figure A20068004753500819
(U.S. Stryker Corp. (Stryker Inc.), MI, USA) or ChronOS
Figure A200680047535008110
(Lin Saisi company (Synthes GmbH), bone Switzerland) is transplanted granule and is immersed in the UCMC cell suspending liquid, and mixing.The UCMC cell that biomaterial is gone in inoculation was kept in the PTT-4 culture medium 48 hours in 37 ℃ in the cell culture incubator, and then it was being carried out the Laser Scanning Confocal Microscope analysis with FDA and propidium iodide dyeing.The UCMC cell inoculation of living is gone into TissuFleece
Figure A200680047535008111
(Figure 30 A) and BoneSave
Figure A200680047535008112
(U.S. Stryker Corp. (Stryker Inc.), MI USA) in (Figure 30 B), are hatched as above-mentioned, use FDA and propidium iodide (PI) dyeing then, carry out the Laser Scanning Confocal Microscope analysis then.The result is presented among Figure 30 A and the 30B.Present redness (in Figure 30 A and 30B, showing) with the painted cell of PI, present green (in Figure 30 A and 30B, showing) with " G " with the painted cell of FDA with " R ".
Fluorescein diacetate (FDA) (50 μ g/ml) and PI (25 μ g/ml) use 1 * PBS preparation.For the dyeing of cell, from incubator, take out them also with 1 * PBS washed twice.Then they and FDA (50 μ g/ml) were hatched 15 minutes in 37 ℃ of incubators.After this, with cell with 1 * PBS washed twice.And then redyed 5 minutes in room temperature with PI (25 μ g/ml).Then, cell is prevented drying with 1 * PBS washed twice and with sample preservation in 1 * PBS.After this, as above-mentioned under Laser Scanning Confocal Microscope observation of cell.
This result of experiment shows that UCMC grows on different types of support.Therefore, UCMC can be used for transforming soft tissue alive and sclerous tissues, is used for repairing and regeneration.
Embodiment 22: the collagen scaffold of UCMC-propagation is implanted in the immunocompetent mice
This experimental results show that the angiogenesis character of UCMC.As in embodiment 12, describe before with cell seeding in support.Figure 31 A has shown the outward appearance at the collagen lattice (support) of external UCMC-propagation.Figure 31 B has shown the collagen lattice that acellular collagen lattice contrasts and UCMC-breeds that implant the 21st day in mice.Do not observe the signal of immunologic rejection.At the 21st day, in the collagen lattice of the UCMC-propagation of implanting, observe macroscopic angiogenesis.Figure 31 C has shown the angiogenesis (being pointed out by arrow) of the microcosmic of the collagen lattice that UCMC-breeds.
This experiment has clearly shown the angiogenesis character of UCMC.Therefore, these cells can be used for strengthening the angiogenesis in tissue repair and regeneration, and are used for the treatment of ischemia sexual disorders.
Embodiment 23:UCMC is used for the clinical practice of FTB treatment
This experiment has shown the treatment to the 53 years old female patient that suffers FTB.Generally speaking, this class burn must be cured fully in skin transplantation.
In order to test, press the mesenchymal cell (UCMC) of the separation umbilical cord amniotic membrane of describing among the embodiment 2.
For this experiment, at first with patient's general anesthesia to be treated, then at Biobrane with UCMC propagation
Figure A20068004753500821
(Tao Shi Rickman pharmaceutical companies (Down HickamPharmaceuticals), Texas USA) is applied to before patient's the wound-L nylon membrane, uses the downright bad tissue (referring to Figure 32 B) of surgery curette surgical removal burn.
In this experiment, with Biobrane
Figure A20068004753500822
-L nylon membrane cuts into the disk that is fit to 32mm tissue culture ware.In 150mm tissue culture ware, cultivate UCMC, scrape results, centrifugal and by the aforesaid counting of this paper by cell.With 100000 cell/cm 2Density be inoculated into UCMC on the film before, with DMEM culture medium moistening diaphragm 48 hours (referring to Figure 32 B, first width of cloth figure).
The wound bed that Figure 32 A is presented on the FTB (3 degree) of 53 years old female patient is prepared.UCMC is inoculated into Biobrane -L wound dressing (Tao Shi Rickman pharmaceutical companies (Down HickamPharmaceuticals), Texas, USA) on.The UCMC-Biobrane construct is transferred to (Figure 32 B) on the wound.Saw healing (not having skin graft) fully at the 7th day, and follow up a case by regular visits to 3 months stable.This situation shows that UCMC can have the healing power of stem cell, does not use skin graft also to cure this type of burn, represents that this technology is at alternative auto-skin grafting in future.
Figure 34 shows the treatment to 2 years old male patient's FTB.Test carry out similar to previous experiments.UCMC is cultivated in DMEM/10%FCS up to converging on tissue culture's ware.Harvesting and and SoloSite then swipe
Figure A20068004753500831
The gel ((Smith﹠amp of brightness company of Xerox; Nephew), Hull UK) mixes, and with 100 ten thousand cell/em 2Density stick with paste on the wound.SoloSite
Figure A20068004753500832
Be the hydrogel wound dressing that antiseptic is arranged, it comprises water-swollen polymer, and it keeps the gel sample up to saturated.It can contribute the tissue of the rehydrated necrosis of moisture.It absorbs exudate and keeps its structure on wound simultaneously.Without skin transplantation, observed the healing completely of this method of use at the 5th day.
Embodiment 24:UCMC is used for the clinical practice of part cortex trauma care
This experiment has shown that UCMC is used for the clinical practice of 2 years old male patient's part cortex wound (2 degree) treatment.Experiment is by the carrying out of describing among the embodiment 23 (wound is prepared and the preparation of wound dressing, etc.).At Tegaderm
Figure A20068004753500833
(3M health care company (3M Health Care), Minnesota USA) go up to cultivate UCMC and it is transferred on the wound in wound dressing.
Tegaderm
Figure A20068004753500834
Wound dressing is quick capillarity, non-bloating polyurethane foam, has the thin film backing (film backing) of highly-breathable, and it prevents that the wound exudate from appearing and being the barrier of outside contamination, and that dressing is kept perfectly is non-leakage.Wound dressing is made up of urethanes thin film backing (5-10wt.-%), polyurethane foam (90-95wt.-%) and small volume of ink (0.1-1wt.-%).
Observed the healing fully (referring to Figure 33) of part cortex wound at the 3rd day.The arrow indicating area A in left side among Figure 33 is promptly with the Tegaderm that UCMC is housed
Figure A20068004753500835
The wound part of wound dressing treatment.The arrow indicating area B on right side, i.e. wound part for the treatment of with conventional method.
Embodiment 25:UCMC is used for the clinical practice of non-healing radiation trauma care
This embodiment shows that UCMC is used for the clinical practice 1 years old non-healing radiation wound of child's treatment, and this child suffers from hemangioma.Use conventional trauma care, initial wound is in the not healing in period that surpasses 90 days.Experiment is by the carrying out of describing among the embodiment 23.UCMC is pressed 100000 cell/cm 2Density cultivate at Tegaderm
Figure A20068004753500841
In the wound dressing, and transfer on the wound for twice weekly.As shown in Figure 35, radiation in the period wound through more than 20 days UCMC cell therapy heals fully.
Embodiment 26:UCMC is used for non-healing diabetic wound and non-healing diabetes food wound Hinder the clinical practice of treatment
These embodiment show that UCMC is used for the treatment of the clinical practice of the flap donor position wound (Figure 37 A) of non-healing diabetic wound (Figure 36), non-healing diabetes traumatism by food (Figure 37 B) and failure.Both healings under the conventional therapy more than 6 years by a definite date of back are failed.
Press to describe among the embodiment 23 and cultivate UCMC, and and SoloSite
Figure A20068004753500842
The gel ((Smith﹠amp of brightness company of Xerox; Nephew), Hull UK) mixes.UCMC/SoloSite
Figure A20068004753500843
Gel mixture is by 100 ten thousand cell/cm 2Density stick with paste on wound.Carry out application once in a week to wound.Observe the healing fully (Figure 36) of non-healing diabetic wound at the 26th day, and under the situation of flap donor position wound (Figure 37 A) of non-healing diabetes traumatism by food (Figure 37 B) and failure, can observe the process of wound healing.
Embodiment 27:UCEC directly is divided into hepatocyte
Press the epithelium ancestral cells (UCEC) of the separation umbilical cord amniotic membrane of describing among the embodiment 2.For UCEC is divided into hepatocyte, (the BBRC06H culture medium is the modification version of the BBRC06 that describes in by embodiment 15, does not add nicotiamide and has added the Oncostatin. of 50 μ g/ml-M) in the BBRC06H culture medium that contains Oncostatin .-M (50 μ g/ml) with cellular exposure.
Figure 38 has shown that the albumin of UCEC is expressed after inducing with the BBRC06H culture medium that contains Oncostatin .-M (50 μ g/ml).Alpha-fetoprotein is the stem cell sign of liver ancestral cells.When inducing with inducing culture, UCEC is divided into the how albuminised ripe hepatocyte of production.Tubulin is a house-keeping gene, is used for showing sample on the equivalent that the Western trace measures.This tests demonstration, and UCEC has and is divided into hepatocellular potentiality, and it can be used for liver disease or the Cytotoxic external model of test new drug.

Claims (97)

1, skin equivalent, it comprises:
Support, it comprises from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane.
2, according to the skin equivalent of claim 1, wherein said is mescenchymal stem cell (UCMC) or epithelial stem cell (UCEC) from the deutero-cell of ancestral cells.
3, according to the skin equivalent of claim 1 or 2, wherein said cell is the cell from body.
4, according to the skin equivalent of claim 1 or 2, wherein said cell is a heterogenous cell.
5, according to the skin equivalent of claim 1 or 2, wherein said cell is allochthonous cell.
6, according to each skin equivalent in the claim 1 to 5, wherein said cell is the mammal source.
7, according to the skin equivalent of claim 6, wherein said cell is that the people originates.
8, according to each skin equivalent in the claim 1 to 7, wherein said support also comprises at least one cell line, and it can be divided into blood vessel or body of gland.
9, skin equivalent according to Claim 8, wherein said support comprise vascular endothelial cell or microvascular dermal endothelial cell (DMEC).
10, according to the skin equivalent of claim 9, wherein said vascular endothelial cell is deutero-from umbilical cord.
11, according to the skin equivalent of claim 10, wherein said cord vessels endotheliocyte is that the people originates.
12, according to each skin equivalent in the claim 1 to 7, wherein said support also comprises the cell of old and feeble keratinocyte.
13, the skin equivalent of each in requiring according to aforesaid right, its medium-height trestle comprises biodegradable material.
14, according to each skin equivalent in the claim 1 to 12, its medium-height trestle comprises material, and described material is selected from agarose, polycaprolactone, niobium bag by carbon, chitosan, collagen, hyaluronic acid, calcium phosphate, starch, hydroxyapatite, fibrin, alginate, polyglycolic acid, carbon nano-fiber, porous Merlon, politef, polylactide and composition thereof.
15, according to the skin equivalent of claim 14, wherein timbering material is the porous Merlon.
16, the skin equivalent of each in requiring according to aforesaid right, wherein said support comprises at least a extracellular matrix component.
17, according to the skin equivalent of claim 16, wherein said extracellular matrix component is selected from collagen, elastin laminin, intercellular adhesion molecule, laminin, heparin, fibronectin, Dan Baijutang, tenascin, FBN1 and composition thereof.
18, according to the skin equivalent of claim 17, wherein said extracellular matrix component is a type i collagen.
19, the skin equivalent of each in requiring according to aforesaid right, wherein said cell can be bred and further be divided into fibroblast and keratinocyte.
20, produce the method for skin equivalent, it comprises:
● support is provided,
● will from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane be placed in the described support or on the support and
● hatch described support in first culture medium, it allows described cell proliferation and further differentiation.
21, according to the method for claim 20, wherein said is mescenchymal stem cell (UCMC) from the deutero-cell of ancestral cells.
22, according to the method for claim 20, wherein said is epithelial stem cell (UCEC) from the deutero-cell of ancestral cells.
23, according to each method in the claim 20 to 22, it also comprises:
● be placed into described cell in the support or before on the support, be placed on extracellular matrix in the described support or on the support.
24, according to the method for claim 21 or 23, wherein method comprises:
● support is provided,
● be placed on UCMC in the described support or on the support,
● hatch described support in first culture medium that is suitable for the fibroblast cultivation, it allows described UCMC propagation and further differentiation,
● be placed on UCEC in the described support or on the support and
● hatch described support in second culture medium, described second culture medium allows described UCEC propagation and further differentiation.
25, according to each method in the claim 20 to 24, wherein said cell is the cell from body.
26, according to each method in the claim 20 to 24, wherein said cell is the cell of allosome.
27, according to each method in the claim 20 to 24, wherein said cell is allochthonous cell.
28, according to each method in the claim 24 to 27, wherein said cell is from mammal.
29, according to the method for claim 28, wherein said cell is from the people.
30, according to each method in the claim 24 to 29, wherein said support also comprises at least one cell line, and it can be divided into blood vessel or body of gland.
31, according to the method for claim 30, wherein said support comprises vascular endothelial cell or microvascular dermal endothelial cell (DMEC).
32, according to the method for claim 31, wherein said vascular endothelial cell is deutero-from umbilical cord.
33, according to the method for claim 32, wherein said cord vessels endotheliocyte is that the people originates.
34, according to the method for claim 21, wherein when described support comprised UCMC, described first culture medium comprised the culture medium that is suitable for the fibroblast cultivation, was fibroblast thereby breed and break up described UCMC.
35, according to the method for claim 24 or 34, wherein said first culture medium comprise the fibroblastic growth culture medium and fetal blood clear, for example tire calf serum (FCS) or hyclone (FBS), thereby propagation and to break up described UCMC be fibroblast.
36, according to the method for claim 35, wherein said fibroblastic growth culture medium is selected from
Figure A2006800475350004C1
-keratinocyte culture medium (Kang Baisi company (Cambrex)), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex)),
Figure A2006800475350004C2
Culture medium (Ka Sikade biotech firm (Cascade Biologics)), Green ' s culture medium, CMRL1066 (Medi-Tech. Inc (Mediatech.Inc)), M171 (Ka Sikade biotech firm (Cascade Biologics)), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.
37, according to each method in the claim 34 to 36, wherein said first culture medium comprise about 90 to about 95% (v/v) fibroblastic growth culture medium and about 5 to about 10% tire cattle or tire calf serum (FCS).
38, according to the method for claim 37, wherein said first culture medium comprises about 90 to about 95% (v/v) CMRL1066 (Medi-Tech. Inc (Mediatech.Inc)) and about 5 to about 10% tire calf serum (FCS).
39, according to the method for claim 22, wherein when described support comprised UCEC, described first culture medium comprised the culture medium that is suitable for the keratinocyte cultivation, was keratinocyte thereby breed and break up described UCEC.
40, according to the method for claim 39, wherein said first culture medium comprises keratinocyte growth medium, somatomedin, insulin, transferrins and Monohydrated selenium dioxide.
41, according to the method for claim 40, wherein said somatomedin is selected from epidermal growth factor (EGF), insulin-like growth factor-i, platelet derived growth factor-BB (PDGFb), transforming growth factor-beta, keratinocyte growth factor (KGF), TGF-α and amphiregulin.
42, according to the method for claim 40 or 41, wherein said keratinocyte growth medium is selected from -keratinocyte culture medium (Kang Baisi company (Cambrex)), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex)),
Figure A2006800475350005C2
Culture medium (Ka Sikade biotech firm (Cascade Biologics)), Green ' s culture medium, CMRL1066 (Medi-Tech. Inc (Mediatech.Inc)), M171 (Ka Sikade biotech firm (Cascade Biologics)), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.
43, according to each method in the claim 39 to 42, wherein said first culture medium comprises keratinocyte growth medium, epidermal growth factor (EGF), insulin, transferrins and Monohydrated selenium dioxide.
44, according to the method for claim 43, wherein said first culture medium comprises Culture medium (Ka Sikade biotech firm (Cascade Biologics)), epidermal growth factor (EGF), insulin, transferrins and Monohydrated selenium dioxide.
45, according to the method for claim 44, wherein said first culture medium comprises about 98.8 to about 99.4% (v/v)
Figure A2006800475350006C1
Culture medium (Ka Sikade biotech firm (Cascade Biologics)), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and about 10ng/ml epidermal growth factor (EGF).
46, according to the method for claim 24, wherein as in the claim 38 to 44 each desired as described in second culture medium comprise and be suitable for the culture medium that keratinocyte is cultivated.
47, according to each method in the claim 20 to 46, its medium-height trestle comprises biodegradable material.
48, according to each method in the claim 20 to 46, its medium-height trestle comprises material, and described material is selected from agarose, polycaprolactone, niobium bag by carbon, chitosan, collagen, hyaluronic acid, calcium phosphate, starch, hydroxyapatite, fibrin, alginate, polyglycolic acid, carbon nano-fiber, porous Merlon, politef, polylactide and composition thereof.
49, according to the method for claim 48, wherein timbering material is the porous Merlon.
50, according to each method in the claim 20 to 49, wherein extracellular matrix component is in described cell is placed on described support or on the described support.
51, according to the method for claim 23 or 50, wherein said extracellular matrix component is selected from collagen, elastin laminin, intercellular adhesion molecule, laminin, heparin, fibronectin, Dan Baijutang, tenascin, FBN1 and composition thereof.
52, according to the method for claim 51, wherein said extracellular matrix component is a type i collagen.
53, the method for treatment dermatosis, it comprises that the skin equivalent with each definition in the claim 1 to 19 is applied in the skin area with described dermatosis.
54, according to the method for claim 53, wherein said dermatosis is skin, radiation wound, diabetic wound or the ulcer of burn.
55, according to the skin equivalent of one of claim 1 to 19 or be used for the purposes of preparation of drug combination according to the skin equivalent that the method for one of claim 20 to 54 obtains.
56, according to the pharmaceutical composition of claim 55, it is used for the treatment of burned skin, ulcer, radiation wound or diabetic wound.
57, the cell bank that comprises the skin equivalent that obtains according to the skin equivalent of one of claim 1 to 19 or according to the method for one of claim 20 to 54.
58, produce the method for mucoprotein-production cell, it comprises:
● umbilical cord amniotic membrane epithelial stem cell (UCEC) or umbilical cord amnion mesenchymal stem cell (UCMC) be placed in the container and
● in the culture medium that is fit to mucoprotein-production cell culture, hatch described cell.
59, according to the method for claim 58, wherein said culture medium comprises growth medium, insulin, transferrins, Monohydrated selenium dioxide and the somatomedin that is used for mucoprotein-production cell culture.
60, according to the method for claim 59, wherein said somatomedin is selected from epidermal growth factor (EGF), insulin-like growth factor-i, platelet derived growth factor-BB (PDGFb), transforming growth factor-beta, keratinocyte growth factor (KGF), TGF-α and amphiregulin.
61, according to the method for claim 59 or 60, the wherein said growth medium that is used for mucoprotein-production cell culture is selected from
Figure A2006800475350007C1
-keratinocyte culture medium (Kang Baisi company (Cambrex)), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex)),
Figure A2006800475350007C2
Culture medium (Ka Sikade biotech firm (Cascade Biologics)), Green ' s culture medium, CMRL1066 (Medi-Tech. Inc (Mediatech.Inc)), M171 (Ka Sikade biotech firm (Cascade Biologics)), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.
62, according to each method in the claim 58 to 61, wherein said be fit to the mucoprotein-culture medium of producing cell culture comprise be used for mucoprotein-produce growth medium, epidermal growth factor (EGF), insulin, transferrins and the Monohydrated selenium dioxide of cell culture.
63, according to the method for claim 62, wherein said be fit to the mucoprotein-culture medium of producing cell culture comprise about 98.8 to about 99.4% (v/v) be used for mucoprotein-produce cell culture growth mediums, about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and about 10ng/ml epidermal growth factor (EGF).
64, according to the method for claim 63, wherein said be fit to the mucoprotein-culture medium of producing cell culture comprise about 98.8 to about 99.4% (v/v) CMRL1066 (Medi-Tech. Inc (Mediatech.Inc)), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and about 10ng/ml epidermal growth factor (EGF).
65, according to the method for claim 63, wherein said be fit to the mucoprotein-culture medium of producing cell culture comprise about 98.8 to about 99.4% (v/v) M171 (Ka Sikade biotech firm (CascadeBiologics)), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrins, about 0.2 to about 0.4% (v/v) Monohydrated selenium dioxide and about 10ng/ml epidermal growth factor (EGF).
66, the method for the cell that influenced by cigarette of treatment, it comprises makes the tissue that comprises the cell that is influenced by cigarette and the mucoprotein production cells contacting that is produced according to each definition in the claim 58 to 65.
67, according to the method for claim 66, the cell that wherein is affected is the cell on respiratory tract or eye surface.
68, treatment synovial cell sarcoma, cigarette suck the method for damage or eye surface damage, and that it uses that each method by claim 58 to 65 obtains is mucoprotein-produce cell.
69, that each the method by claim 58 to 65 obtains is mucoprotein-produce the purposes of cell, and it is used for esophagus and air flue organizational project, is used for cosmetic applications or as genes matter delivery system.
70, produce the method for insulin-production cell, it comprises:
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane and
● in proper culture medium propagation with break up described cell and become β-islet cells.
71, according to the method for claim 70, it also comprises
● separate the insulin of producing by described β-islet cells.
72, according to the method for claim 70 or 71, wherein said is mescenchymal stem cell (UCMC) or epithelial stem cell (UCEC) from the deutero-cell of ancestral cells.
73, according to each method of claim 70 to 72, wherein said proper culture medium comprises nicotiamide.
74, according to the method for claim 73, wherein said culture medium also comprises and is used for the growth medium that β-islet cells is cultivated.
75, according to the method for claim 74, the fetal blood that wherein said culture medium also comprises somatomedin or calf or Niu Laiyuan is clear.
76, according to the method for claim 74 or 75, wherein said culture medium also comprises insulin, transferrins and Monohydrated selenium dioxide.
77, according to the method for claim 75 or 76, wherein said somatomedin is selected from epidermal growth factor (EGF), insulin-like growth factor-i, platelet derived growth factor-BB (PDGFb), transforming growth factor-beta, keratinocyte growth factor (KGF), TGF-α and amphiregulin.
78, according to each method of claim 74 to 77, the growth medium that the wherein said β of being used for-islet cells is cultivated is selected from
Figure A2006800475350009C1
-keratinocyte culture medium (Kang Baisi company (Cambrex)), MEGM-breast epithelial cell culture medium (Kang Baisi company (Cambrex)),
Figure A2006800475350009C2
Culture medium (Ka Sikade biotech firm (Cascade Biologics)), Green ' s culture medium, CMRL1066 (Medi-Tech. Inc (Mediatech.Inc)), M171 (Ka Sikade biotech firm (Cascade Biologics)), L-15 culture medium, Dulbecco ' s improve Eagle culture medium (DMEM), DMEM-F12 and RPMI culture medium.
79, the insulin production cell that obtains of the method for each definition by claim 70 to 78.
80, the method for the unbalance relevant disease of treatment and insulin level, it comprises the insulin production cell that obtains to the method for administration by each definition of claim 70 to 78.
81, the method for claim 80, wherein disease is insulin-dependent diabetes (IDDM).
82, the purposes of the insulin of producing according to each method of claim 71 to 78, it is used for the treatment of insulin-dependent diabetes (IDDM).
83, the method for treatment osteopathia, it comprises uses the osteoblast that produces from the isolating mescenchymal stem cell of umbilical cord amniotic membrane (UCMC).
84, the method for treatment cartilage disease, it comprises uses the chondrocyte of producing from the isolating mescenchymal stem cell of umbilical cord amniotic membrane.
85, produce the method that dopamine and tyrosine hydroxylase are produced cell, it comprises
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane and
● in proper culture medium propagation with break up described cell and become dopamine and tyrosine hydroxylase to produce cell.
86,5 method according to Claim 8, it also comprises
● separate dopamine and tyrosine hydroxylase by described dopamine and tyrosine hydroxylase production cells produce.
87,5 or 86 method according to Claim 8, wherein said is mescenchymal stem cell (UCMC) from the deutero-cell of ancestral cells.
88, produce the method that human leucocyte antigen (HLA) G (HLA-G) produces cell, it comprises
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane and
● in proper culture medium propagation with break up described cell and become HLA-G to produce cell.
89,8 method according to Claim 8, it also comprises
● separate described HLA-G and produce the HLA-G that cell produces.
90,8 or 89 method according to Claim 8, wherein said is mescenchymal stem cell (UCMC) or epithelial stem cell (UCEC) from the deutero-cell of ancestral cells.
91, induce the method for old and feeble keratinocyte propagation, it comprises
● in suitable growth medium, cultivate old and feeble keratinocyte and
● the mescenchymal stem cell (UCMC) of umbilical cord amniotic membrane is added in the keratinocyte of described aging, to induce the keratinocyte propagation of described aging.
92, according to the method for claim 90, wherein said method also comprises
● separate described propagation aging keratinocyte and
● be applied to the keratinocyte of the aging of described propagation in the support or on the support.
93, according to each method of claim 91 or 92, the keratinocyte of wherein said aging is isolating in 35 years old or above, 40 years old or above, 50 years old or above, 60 years old or above or 70 years old or above patient.
94, produce hepatocellular method, it comprises
● cultivate from the deutero-cell of the isolating ancestral cells of umbilical cord amniotic membrane and
● in proper culture medium propagation with break up described cell and become hepatocyte.
95, according to the method for claim 94, wherein said is epithelial stem cell (UCEC) from the deutero-cell of ancestral cells.
96, according to the method for claim 94 or 95, wherein said proper culture medium comprises Oncostatin .-M.
97, according to the method for claim 96, wherein said culture medium also comprises the growth medium that is used for liver cell culture.
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